Nagata Y

References (56)

Title : Structural and catalytic effects of surface loop-helix transplantation within haloalkane dehalogenase family - Marek_2020_Comput.Struct.Biotechnol.J_18_1352
Author(s) : Marek M , Chaloupkova R , Prudnikova T , Sato Y , Rezacova P , Nagata Y , Kuta Smatanova I , Damborsky J
Ref : Comput Struct Biotechnol J , 18 :1352 , 2020
Abstract : Engineering enzyme catalytic properties is important for basic research as well as for biotechnological applications. We have previously shown that the reshaping of enzyme access tunnels via the deletion of a short surface loop element may yield a haloalkane dehalogenase variant with markedly modified substrate specificity and enantioselectivity. Here, we conversely probed the effects of surface loop-helix transplantation from one enzyme to another within the enzyme family of haloalkane dehalogenases. Precisely, we transplanted a nine-residue long extension of L9 loop and beta4 helix from DbjA into the corresponding site of DbeA. Biophysical characterization showed that this fragment transplantation did not affect the overall protein fold or oligomeric state, but lowered protein stability (DeltaT (m) = -5 to 6 degC). Interestingly, the crystal structure of DbeA mutant revealed the unique structural features of enzyme access tunnels, which are known determinants of catalytic properties for this enzyme family. Biochemical data confirmed that insertion increased activity of DbeA with various halogenated substrates and altered its enantioselectivity with several linear beta-bromoalkanes. Our findings support a protein engineering strategy employing surface loop-helix transplantation for construction of novel protein catalysts with modified catalytic properties.
ESTHER : Marek_2020_Comput.Struct.Biotechnol.J_18_1352
PubMedSearch : Marek_2020_Comput.Struct.Biotechnol.J_18_1352
PubMedID: 32612758
Gene_locus related to this paper: brael-e2rv62

Title : Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome - Anda_2015_Proc.Natl.Acad.Sci.U.S.A_112_14343
Author(s) : Anda M , Ohtsubo Y , Okubo T , Sugawara M , Nagata Y , Tsuda M , Minamisawa K , Mitsui H
Ref : Proc Natl Acad Sci U S A , 112 :14343 , 2015
Abstract : rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.
ESTHER : Anda_2015_Proc.Natl.Acad.Sci.U.S.A_112_14343
PubMedSearch : Anda_2015_Proc.Natl.Acad.Sci.U.S.A_112_14343
PubMedID: 26534993
Gene_locus related to this paper: 9rhiz-a0a0s2en26 , 9rhiz-a0a0p0z830 , 9rhiz-a0a0p0z878 , 9rhiz-a0a0p0z9k0 , 9rhiz-a0a0s2epz8

Title : Complete Genome Sequence of Polypropylene Glycol- and Polyethylene Glycol-Degrading Sphingopyxis macrogoltabida Strain EY-1 - Ohtsubo_2015_Genome.Announc_3_e01399
Author(s) : Ohtsubo Y , Nagata Y , Numata M , Tsuchikane K , Hosoyama A , Yamazoe A , Tsuda M , Fujita N , Kawai F
Ref : Genome Announc , 3 : , 2015
Abstract : Strain EY-1 was isolated from a microbial consortium growing on a random polymer of ethylene oxide and propylene oxide. Strain EY-1 grew on polyethylene glycol and polypropylene glycol and identified as Sphingopyxis macrogoltabida. Here, we report the complete genome sequence of Sphingopyxis macrogoltabida EY-1. The genome of strain EY-1 is comprised of a 4.76-Mb circular chromosome, and five plasmids. The whole finishing was conducted in silico, with aids of computational tools GenoFinisher and AceFileViewer. Strain EY-1 is available from Biological Resource Center, National Institute of Technology and Evaluation (Tokyo, Japan) (NITE).
ESTHER : Ohtsubo_2015_Genome.Announc_3_e01399
PubMedSearch : Ohtsubo_2015_Genome.Announc_3_e01399
PubMedID: 26634754
Gene_locus related to this paper: sphmc-a0a0n9ujk2

Title : Properties and biotechnological applications of natural and engineered haloalkane dehalogenases - Nagata_2015_Appl.Microbiol.Biotechnol_99_9865
Author(s) : Nagata Y , Ohtsubo Y , Tsuda M
Ref : Applied Microbiology & Biotechnology , 99 :9865 , 2015
Abstract : Haloalkane dehalogenases (HLDs) convert halogenated compounds to corresponding alcohols, halides, and protons. They belong to alpha/beta-hydrolases, and their principal catalytic mechanism is SN2 nucleophilic substitution followed by the addition of water. Since HLDs generally have broad and different substrate specificities, they have various biotechnological applications. HLDs have previously been believed to be present only in bacterial strains that utilize xenobiotic halogenated compounds, and three archetypal HLDs, i.e., DhlA, DhaA, and LinB, have been intensively investigated by biochemical, structural, and computational analyses. Furthermore, by using the resulting data and target-selected random mutagenesis approaches, these HLDs have been successfully engineered to improve their substrate specificities and activities. In addition, important insights into protein evolution have been obtained by studying these HLDs. At the same time, the genome and metagenome information has revealed that HLD homologues are widely distributed in many bacterial strains, including ones that have not been reported to degrade halogenated compounds. Some of these cryptic HLD homologues have been experimentally confirmed to be "true" HLDs with unique substrate specificities and enantioselectivities. Although their biological functions and physiological roles remain mysterious, these potential HLDs are considered promising materials for the development of new biocatalysts.
ESTHER : Nagata_2015_Appl.Microbiol.Biotechnol_99_9865
PubMedSearch : Nagata_2015_Appl.Microbiol.Biotechnol_99_9865
PubMedID: 26373728

Title : Complete Genome Sequence of a Phenanthrene Degrader, Mycobacterium sp. Strain EPa45 (NBRC 110737), Isolated from a Phenanthrene-Degrading Consortium - Kato_2015_Genome.Announc_3_e00782
Author(s) : Kato H , Ogawa N , Ohtsubo Y , Oshima K , Toyoda A , Mori H , Nagata Y , Kurokawa K , Hattori M , Fujiyama A , Tsuda M
Ref : Genome Announc , 3 : , 2015
Abstract : A phenanthrene degrader, Mycobacterium sp. EPa45, was isolated from a phenanthrene-degrading consortium. Here, we report the complete genome sequence of EPa45, which has a 6.2-Mb single circular chromosome. We propose a phenanthrene degradation pathway in EPa45 based on the complete genome sequence.
ESTHER : Kato_2015_Genome.Announc_3_e00782
PubMedSearch : Kato_2015_Genome.Announc_3_e00782
PubMedID: 26184940
Gene_locus related to this paper: 9myco-a0a0g3iim6 , 9myco-a0a0g3ijm3 , 9myco-a0a0g3ipv9 , 9myco-a0a0g3iuf8 , 9myco-a0a0g3ivy5 , 9myco-a0a0g3ihb1

Title : Complete Genome Sequence of Polyvinyl Alcohol-Degrading Strain Sphingopyxis sp. 113P3 (NBRC 111507) - Ohtsubo_2015_Genome.Announc_3_e01169
Author(s) : Ohtsubo Y , Nagata Y , Numata M , Tsuchikane K , Hosoyama A , Yamazoe A , Tsuda M , Fujita N , Kawai F
Ref : Genome Announc , 3 :e01169 , 2015
Abstract : Strain 113P3 was isolated from activated sludge and identified as a polyvinyl alcohol (PVA)-degrading Pseudomonas species; it was later reidentified as Sphingopyxis species. Only three genes are directly relevant to the metabolism of PVA and comprise the pva operon, which was deposited as accession no. AB190228. Here, we report the complete genome sequence of strain 113P3, which has been conserved as a stock culture (NBRC 111507) at the Biological Resource Center, National Institute of Technology and Evaluation (NITE) (Tokyo, Japan). The genome of strain 113P3 is composed of a 4.4-Mb circular chromosome and a 243-kb plasmid. The whole finishing was conducted in silico except for four PCRs. The sequence corresponding to AB190288 exists on the chromosome.
ESTHER : Ohtsubo_2015_Genome.Announc_3_e01169
PubMedSearch : Ohtsubo_2015_Genome.Announc_3_e01169
PubMedID: 26472829
Gene_locus related to this paper: sphs1-a0a0m4cze0

Title : Complete Genome Sequence of the Thermophilic Polychlorinated Biphenyl Degrader Geobacillus sp. Strain JF8 (NBRC 109937) - Shintani_2014_Genome.Announc_2_e01213
Author(s) : Shintani M , Ohtsubo Y , Fukuda K , Hosoyama A , Ohji S , Yamazoe A , Fujita N , Nagata Y , Tsuda M , Hatta T , Kimbara K
Ref : Genome Announc , 2 : , 2014
Abstract : Geobacillus sp. strain JF8 (NBRC 109937) utilizes biphenyl and naphthalene as sole carbon sources and degrades polychlorinated biphenyl (PCB) at 60 degrees C. Here, we report the complete nucleotide sequence of the JF8 genome (a 3,446,630-bp chromosome and a 39,678-bp plasmid). JF8 has the smallest genome among the known PCB degraders.
ESTHER : Shintani_2014_Genome.Announc_2_e01213
PubMedSearch : Shintani_2014_Genome.Announc_2_e01213
PubMedID: 24459274
Gene_locus related to this paper: geotn-a4isp0 , 9baci-s5yvc0 , 9baci-s5yzj8

Title : Complete Genome Sequence of Pseudomonas aeruginosa MTB-1, Isolated from a Microbial Community Enriched by the Technical Formulation of Hexachlorocyclohexane - Ohtsubo_2014_Genome.Announc_2_e01130
Author(s) : Ohtsubo Y , Sato T , Kishida K , Tabata M , Ogura Y , Hayashi T , Tsuda M , Nagata Y
Ref : Genome Announc , 2 : , 2014
Abstract : Pseudomonas aeruginosa MTB-1 does not degrade gamma-hexachlorocyclohexane (gamma-HCH), but this bacterium persistently coexists with a gamma-HCH-degrading strain, Sphingomonas sp. MM-1, in a microbial community enriched by the technical formulation of HCH. Here we report the complete MTB-1 genome sequence, with a 6.6-Mb circular chromosome.
ESTHER : Ohtsubo_2014_Genome.Announc_2_e01130
PubMedSearch : Ohtsubo_2014_Genome.Announc_2_e01130
PubMedID: 24459257
Gene_locus related to this paper: pseae-PA3695 , pseae-PA5080 , pseae-q9i252

Title : Structural and functional analysis of a novel haloalkane dehalogenase with two halide-binding sites - Chaloupkova_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_1884
Author(s) : Chaloupkova R , Prudnikova T , Rezacova P , Prokop Z , Koudelakova T , Daniel L , Brezovsky J , Ikeda-Ohtsubo W4 , Sato Y , Kuty M , Nagata Y , Kuta Smatanova I , Damborsky J
Ref : Acta Crystallographica D Biol Crystallogr , 70 :1884 , 2014
Abstract : The crystal structure of the novel haloalkane dehalogenase DbeA from Bradyrhizobium elkanii USDA94 revealed the presence of two chloride ions buried in the protein interior. The first halide-binding site is involved in substrate binding and is present in all structurally characterized haloalkane dehalogenases. The second halide-binding site is unique to DbeA. To elucidate the role of the second halide-binding site in enzyme functionality, a two-point mutant lacking this site was constructed and characterized. These substitutions resulted in a shift in the substrate-specificity class and were accompanied by a decrease in enzyme activity, stability and the elimination of substrate inhibition. The changes in enzyme catalytic activity were attributed to deceleration of the rate-limiting hydrolytic step mediated by the lower basicity of the catalytic histidine.
ESTHER : Chaloupkova_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_1884
PubMedSearch : Chaloupkova_2014_Acta.Crystallogr.D.Biol.Crystallogr_70_1884
PubMedID: 25004965
Gene_locus related to this paper: brael-e2rv62

Title : Stepwise enhancement of catalytic performance of haloalkane dehalogenase LinB towards beta-hexachlorocyclohexane - Moriuchi_2014_AMB.Express_4_72
Author(s) : Moriuchi R , Tanaka H , Nikawadori Y , Ishitsuka M , Ito M , Ohtsubo Y , Tsuda M , Damborsky J , Prokop Z , Nagata Y
Ref : AMB Express , 4 :72 , 2014
Abstract : Two haloalkane dehalogenases, LinBUT and LinBMI, each with 296 amino acid residues, exhibit only seven amino acid residue differences between them, but LinBMI's catalytic performance towards beta-hexachlorocyclohexane (beta-HCH) is considerably higher than LinBUT's. To elucidate the molecular basis governing this difference, intermediate mutants between LinBUT and LinBMI were constructed and kinetically characterized. The activities of LinBUT-based mutants gradually increased by cumulative mutations into LinBUT, and the effects of the individual amino acid substitutions depended on combination with other mutations. These results indicated that LinBUT's beta-HCH degradation activity can be enhanced in a stepwise manner by the accumulation of point mutations.
ESTHER : Moriuchi_2014_AMB.Express_4_72
PubMedSearch : Moriuchi_2014_AMB.Express_4_72
PubMedID: 25401073

Title : Complete Genome Sequence of Pseudomonas sp. Strain TKP, Isolated from a gamma-Hexachlorocyclohexane-Degrading Mixed Culture - Ohtsubo_2014_Genome.Announc_2_e01241
Author(s) : Ohtsubo Y , Kishida K , Sato T , Tabata M , Kawasumi T , Ogura Y , Hayashi T , Tsuda M , Nagata Y
Ref : Genome Announc , 2 : , 2014
Abstract : Pseudomonas sp. strain TKP does not degrade gamma-hexachlorocyclohexane (gamma-HCH), but it persistently coexists with the gamma-HCH-degrading Sphingobium sp. strain TKS in a mixed culture enriched by gamma-HCH. Here, we report the complete genome sequence of strain TKP, which consists of one circular chromosome with a size of 7 Mb.
ESTHER : Ohtsubo_2014_Genome.Announc_2_e01241
PubMedSearch : Ohtsubo_2014_Genome.Announc_2_e01241
PubMedID: 24482516
Gene_locus related to this paper: psefl-e2xn15 , psefs-c3k632 , psefs-laaa , psefs-c3k813 , psefl-e2xkc8 , 9psed-v9qxq1 , 9psed-v9qr47

Title : Complete Genome Sequence of the gamma-Hexachlorocyclohexane-Degrading Bacterium Sphingomonas sp. Strain MM-1 - Tabata_2013_Genome.Announc_1_e00247
Author(s) : Tabata M , Ohtsubo Y , Ohhata S , Tsuda M , Nagata Y
Ref : Genome Announc , 1 : , 2013
Abstract : gamma-Hexachlorocyclohexane (gamma-HCH) is a man-made chlorinated insecticide that has caused serious environmental problems. Here, we report the complete genome sequence of the gamma-HCH-degrading bacterium Sphingomonas sp. strain MM-1, which consists of one chromosome and five plasmids. All the specific lin genes that are almost identical to those of Sphingobium japonicum UT26 for the conversion of gamma-HCH to beta-ketoadipate are dispersed on four out of the five plasmids.
ESTHER : Tabata_2013_Genome.Announc_1_e00247
PubMedSearch : Tabata_2013_Genome.Announc_1_e00247
PubMedID: 23682148
Gene_locus related to this paper: 9sphn-m4s0a2 , 9sphn-m4sjq0 , 9sphn-m4s4n0 , 9sphn-m4rwk9 , 9sphn-m4s1q7

Title : Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205 - Okai_2013_J.Bacteriol_195_2642
Author(s) : Okai M , Ohtsuka J , Imai LF , Mase T , Moriuchi R , Tsuda M , Nagata K , Nagata Y , Tanokura M
Ref : Journal of Bacteriology , 195 :2642 , 2013
Abstract : The enzymes LinBUT and LinBMI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of beta-hexachlorocyclohexane (beta-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinBMI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinBMI were categorized into three groups based on the efficiency of the first-step (from beta-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinBMI and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinBMI. The dynamics simulation analyses of wild-type LinBMI and LinBUT revealed that the entrance of the substrate access tunnel of LinBUT was more flexible than that of LinBMI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.
ESTHER : Okai_2013_J.Bacteriol_195_2642
PubMedSearch : Okai_2013_J.Bacteriol_195_2642
PubMedID: 23564170
Gene_locus related to this paper: sphpi-q6vqx3

Title : The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 - Hasan_2013_FEBS.J_280_3149
Author(s) : Hasan K , Gora A , Brezovsky J , Chaloupkova R , Moskalikova H , Fortova A , Nagata Y , Damborsky J , Prokop Z
Ref : Febs J , 280 :3149 , 2013
Abstract : Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K(0.5) for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.
ESTHER : Hasan_2013_FEBS.J_280_3149
PubMedSearch : Hasan_2013_FEBS.J_280_3149
PubMedID: 23490078

Title : Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 - Mase_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_68_652
Author(s) : Mase T , Yabuki H , Okai M , Ohtsuka J , Imai FL , Nagata Y , Tanokura M
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 68 :652 , 2012
Abstract : Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 A resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 A. The crystal contained two molecules in the asymmetric unit.
ESTHER : Mase_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_68_652
PubMedSearch : Mase_2012_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_68_652
PubMedID: 22684062

Title : Complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl degrader - Ohtsubo_2012_J.Bacteriol_194_6970
Author(s) : Ohtsubo Y , Maruyama F , Mitsui H , Nagata Y , Tsuda M
Ref : Journal of Bacteriology , 194 :6970 , 2012
Abstract : We report the complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl-degrading strain isolated from a soil sample in Tokyo. The genome contains a single circular 5,196,935-bp chromosome and no plasmids.
ESTHER : Ohtsubo_2012_J.Bacteriol_194_6970
PubMedSearch : Ohtsubo_2012_J.Bacteriol_194_6970
PubMedID: 23209225
Gene_locus related to this paper: 9burk-k0hvd1 , 9burk-k0hw51 , 9burk-k0hxu4 , 9burk-k0i5a9 , 9burk-k0i8t8 , 9burk-k0i3l8 , 9burk-k0i392

Title : Biochemical characteristics of the novel haloalkane dehalogenase DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58 - Hasan_2011_Appl.Environ.Microbiol_77_1881
Author(s) : Hasan K , Fortova A , Koudelakova T , Chaloupkova R , Ishitsuka M , Nagata Y , Damborsky J , Prokop Z
Ref : Applied Environmental Microbiology , 77 :1881 , 2011
Abstract : We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.
ESTHER : Hasan_2011_Appl.Environ.Microbiol_77_1881
PubMedSearch : Hasan_2011_Appl.Environ.Microbiol_77_1881
PubMedID: 21193677

Title : The lin genes for gamma-hexachlorocyclohexane degradation in Sphingomonas sp. MM-1 proved to be dispersed across multiple plasmids - Tabata_2011_Biosci.Biotechnol.Biochem_75_466
Author(s) : Tabata M , Endo R , Ito M , Ohtsubo Y , Kumar A , Tsuda M , Nagata Y
Ref : Biosci Biotechnol Biochem , 75 :466 , 2011
Abstract : A gamma-hexachlorocyclohexane (HCH)-degrading bacterium, Sphingomonas sp. MM-1, was isolated from soil contaminated with HCH isomers. Cultivation of MM-1 in the presence of gamma-HCH led to the detection of five gamma-HCH metabolites, gamma-pentachlorocyclohexene, 2,5-dichloro-2,5-cyclohexadiene-1,4-diol, 2,5-dichlorohydroquinone, 1,2,4-trichlorobenzene, and 2,5-dichlorophenol, strongly suggesting that MM-1 has the lin genes for gamma-HCH degradation originally identified in the well-studied gamma-HCH-degrading strain Sphingobium japonicum UT26. Southern blot, PCR amplification, and sequencing analyses indicated that MM-1 has seven lin genes for the conversion of gamma-HCH to beta-ketoadipate (six structural genes, linA to linF, and one regulatory gene, linR). MM-1 carried four plasmids, of 200, 50, 40, and 30 kb. Southern blot analysis revealed that all seven lin genes were dispersed across three of the four plasmids, and that IS6100, often found close to the lin genes, was present on all four plasmids.
ESTHER : Tabata_2011_Biosci.Biotechnol.Biochem_75_466
PubMedSearch : Tabata_2011_Biosci.Biotechnol.Biochem_75_466
PubMedID: 21389627
Gene_locus related to this paper: sphpi-linb

Title : Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature - Chaloupkova_2011_FEBS.J_278_2728
Author(s) : Chaloupkova R , Prokop Z , Sato Y , Nagata Y , Damborsky J
Ref : Febs J , 278 :2728 , 2011
Abstract : The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 +/- 0.2 degrees C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 degrees C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Delta(R-S)DeltaH and Delta(R-S)DeltaS were determined. The thermodynamic analysis revealed that the resolution of beta-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of alpha-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase.
ESTHER : Chaloupkova_2011_FEBS.J_278_2728
PubMedSearch : Chaloupkova_2011_FEBS.J_278_2728
PubMedID: 21635695

Title : Complete nucleotide sequence of TOL plasmid pDK1 provides evidence for evolutionary history of IncP-7 catabolic plasmids - Yano_2010_J.Bacteriol_192_4337
Author(s) : Yano H , Miyakoshi M , Ohshima K , Tabata M , Nagata Y , Hattori M , Tsuda M
Ref : Journal of Bacteriology , 192 :4337 , 2010
Abstract : To understand the mechanisms for structural diversification of Pseudomonas-derived toluene-catabolic (TOL) plasmids, the complete sequence of a self-transmissible plasmid pDK1 with a size of 128,921 bp from Pseudomonas putida HS1 was determined. Comparative analysis revealed that (i) pDK1 consisted of a 75.6-kb IncP-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pDK1 were highly similar to those of MOB(H) group of mobilizable plasmids, and (iii) the toluene-catabolic (xyl) gene clusters of pDK1 were derived through homologous recombination, transposition, and site-specific recombination from the xyl gene clusters homologous to another TOL plasmid, pWW53. The minireplicons of pDK1 and its related IncP-7 plasmids, pWW53 and pCAR1, that contain replication and partition genes were maintained in all of six Pseudomonas strains tested, but not in alpha- or betaproteobacterial strains. The recipient host range of conjugative transfer of pDK1 was, however, limited to two Pseudomonas strains. These results indicate that IncP-7 plasmids are essentially narrow-host-range and self-transmissible plasmids that encode MOB(H) group-related transfer functions and that the host range of IncP-7-specified conjugative transfer was, unlike the situation in other well-known plasmids, narrower than that of its replication.
ESTHER : Yano_2010_J.Bacteriol_192_4337
PubMedSearch : Yano_2010_J.Bacteriol_192_4337
PubMedID: 20581207

Title : Enantioselectivity of haloalkane dehalogenases and its modulation by surface loop engineering -
Author(s) : Prokop Z , Sato Y , Brezovsky J , Mozga T , Chaloupkova R , Koudelakova T , Jerabek P , Stepankova V , Natsume R , van Leeuwen JG , Janssen DB , Florian J , Nagata Y , Senda T , Damborsky J
Ref : Angew Chem Int Ed Engl , 49 :6111 , 2010
PubMedID: 20645368
Gene_locus related to this paper: braja-dhaa

Title : Complete genome sequence of the representative gamma-hexachlorocyclohexane-degrading bacterium Sphingobium japonicum UT26 - Nagata_2010_J.Bacteriol_192_5852
Author(s) : Nagata Y , Ohtsubo Y , Endo R , Ichikawa N , Ankai A , Oguchi A , Fukui S , Fujita N , Tsuda M
Ref : Journal of Bacteriology , 192 :5852 , 2010
Abstract : Sphingobium japonicum strain UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH), a man-made chlorinated pesticide that causes serious environmental problems due to its toxicity and long persistence, as a sole source of carbon and energy. Here, we report the complete genome sequence of UT26, which consists of two chromosomes and three plasmids. The 15 lin genes involved in gamma-HCH degradation are dispersed on the two chromosomes and one of the three plasmids.
ESTHER : Nagata_2010_J.Bacteriol_192_5852
PubMedSearch : Nagata_2010_J.Bacteriol_192_5852
PubMedID: 20817768
Gene_locus related to this paper: psepa-q6vpf3 , sphju-d4z1p3 , sphju-d4z3u6 , sphpi-linb , sphju-d4yyy8

Title : Crystallization and preliminary X-ray analysis of a novel haloalkane dehalogenase DbeA from Bradyrhizobium elkani USDA94 - Prudnikova_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_353
Author(s) : Prudnikova T , Mozga T , Rezacova P , Chaloupkova R , Sato Y , Nagata Y , Brynda J , Kuty M , Damborsky J , Smatanova IK
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 65 :353 , 2009
Abstract : A novel enzyme, DbeA, belonging to the haloalkane dehalogenase family (EC was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzyme from B. japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. In order to understand the altered activity and specificity of DbeA, its mutant variant DbeA1, which carries the unique fragment of DbjA, was also constructed. Both wild-type DbeA and DbeA1 were crystallized using the sitting-drop vapour-diffusion method. The crystals of DbeA belonged to the primitive orthorhombic space group P2(1)2(1)2(1), while the crystals of DbeA1 belonged to the monoclinic space group C2. Diffraction data were collected to 2.2 A resolution for both DbeA and DbeA1 crystals.
ESTHER : Prudnikova_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_353
PubMedSearch : Prudnikova_2009_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_65_353
PubMedID: 19342778
Gene_locus related to this paper: brael-e2rv62

Title : Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate - Pavlova_2009_Nat.Chem.Biol_5_727
Author(s) : Pavlova M , Klvana M , Prokop Z , Chaloupkova R , Banas P , Otyepka M , Wade RC , Tsuda M , Nagata Y , Damborsky J
Ref : Nat Chemical Biology , 5 :727 , 2009
Abstract : Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.
ESTHER : Pavlova_2009_Nat.Chem.Biol_5_727
PubMedSearch : Pavlova_2009_Nat.Chem.Biol_5_727
PubMedID: 19701186

Title : Insertion sequence-based cassette PCR: cultivation-independent isolation of gamma-hexachlorocyclohexane-degrading genes from soil DNA - Fuchu_2008_Appl.Microbiol.Biotechnol_79_627
Author(s) : Fuchu G , Ohtsubo Y , Ito M , Miyazaki R , Ono A , Nagata Y , Tsuda M
Ref : Applied Microbiology & Biotechnology , 79 :627 , 2008
Abstract : gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.
ESTHER : Fuchu_2008_Appl.Microbiol.Biotechnol_79_627
PubMedSearch : Fuchu_2008_Appl.Microbiol.Biotechnol_79_627
PubMedID: 18425509
Gene_locus related to this paper: psepa-q6vpf3 , sphpi-linb

Title : The identification of catalytic pentad in the haloalkane dehalogenase DhmA from Mycobacterium avium N85: reaction mechanism and molecular evolution - Pavlova_2007_J.Struct.Biol_157_384
Author(s) : Pavlova M , Klvana M , Jesenska A , Prokop Z , Konecna H , Sato T , Tsuda M , Nagata Y , Damborsky J
Ref : J Struct Biol , 157 :384 , 2007
Abstract : Haloalkane dehalogenase DhmA from Mycobacterium avium N85 showed poor expression and low stability when produced in Escherichia coli. Here, we present expression DhmA in newly constructed pK4RP rhodococcal expression system in a soluble and stable form. Site-directed mutagenesis was used for the identification of a catalytic pentad, which makes up the reaction machinery of all currently known haloalkane dehalogenases. The putative catalytic triad Asp123, His279, Asp250 and the first halide-stabilizing residue Trp124 were deduced from sequence comparisons. The second stabilizing residue Trp164 was predicted from a homology model. Five point mutants in the catalytic pentad were constructed, tested for activity and were found inactive. A two-step reaction mechanism was proposed for DhmA. Evolution of different types of catalytic pentads and molecular adaptation towards the synthetic substrate 1,2-dichloroethane within the protein family is discussed.
ESTHER : Pavlova_2007_J.Struct.Biol_157_384
PubMedSearch : Pavlova_2007_J.Struct.Biol_157_384
PubMedID: 17084094

Title : Degradation of beta-hexachlorocyclohexane by haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205 - Ito_2007_Arch.Microbiol_188_313
Author(s) : Ito M , Prokop Z , Klvana M , Otsubo Y , Tsuda M , Damborsky J , Nagata Y
Ref : Arch Microbiol , 188 :313 , 2007
Abstract : The technical formulation of hexachlorocyclohexane (HCH) mainly consists of the insecticidal gamma-isomer and noninsecticidal alpha-, beta-, and delta-isomers, among which beta-HCH is the most recalcitrant and has caused serious environmental problems. A gamma-HCH-utilizing bacterial strain, Sphingobium sp. MI1205, was isolated from soil which had been contaminated with HCH isomers. This strain degraded beta-HCH more rapidly than the well-characterized gamma-HCH-utilizing strain Sphingobium japonicum UT26. In MI1205, beta-HCH was converted to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) via 2,3,4,5,6-pentachlorocyclohexanol (PCHL). A haloalkane dehalogenase LinB (LinB(MI)) that is 98% identical (seven amino-acid differences among 296 amino acids) to LinB from UT26 (LinB(UT)) was identified as an enzyme responsible for the two-step conversion of beta-HCH to TCDL. This property of LinB(MI) contrasted with that of LinB(UT), which catalyzed only the first step conversion of beta-HCH to PCHL. Site-directed mutagenesis and computer modeling suggested that two of the seven different amino acid residues (V134 and H247) forming a catalytic pocket of LinB are important for the binding of PCHL in an orientation suitable for the reaction in LinB(MI). However, mutagenesis also indicated the involvement of other residues for the activity unique to LinB(MI). Sequence analysis revealed that MI1205 possesses the IS6100-flanked cluster that contains two copies of the linB (MI) gene. This cluster is identical to the one located on the exogenously isolated plasmid pLB1, suggesting that MI1205 had recruited the linB genes by a horizontal transfer event.
ESTHER : Ito_2007_Arch.Microbiol_188_313
PubMedSearch : Ito_2007_Arch.Microbiol_188_313
PubMedID: 17516046
Gene_locus related to this paper: sphpi-linb

Title : Weak activity of haloalkane dehalogenase LinB with 1,2,3-trichloropropane revealed by X-Ray crystallography and microcalorimetry - Monincova_2007_Appl.Environ.Microbiol_73_2005
Author(s) : Monincova M , Prokop Z , Vevodova J , Nagata Y , Damborsky J
Ref : Applied Environmental Microbiology , 73 :2005 , 2007
Abstract : 1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k(cat) = 0.005 s(-1)) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.
ESTHER : Monincova_2007_Appl.Environ.Microbiol_73_2005
PubMedSearch : Monincova_2007_Appl.Environ.Microbiol_73_2005
PubMedID: 17259360
Gene_locus related to this paper: sphpi-linb

Title : Complete sequence determination combined with analysis of transposition\/site-specific recombination events to explain genetic organization of IncP-7 TOL plasmid pWW53 and related mobile genetic elements - Yano_2007_J.Mol.Biol_369_11
Author(s) : Yano H , Garruto CE , Sota M , Ohtsubo Y , Nagata Y , Zylstra GJ , Williams PA , Tsuda M
Ref : Journal of Molecular Biology , 369 :11 , 2007
Abstract : Recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (TOL) plasmids and their residing transposons. To elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pWW53, a TOL plasmid from Pseudomonas putida MT53. pWW53 was found to belong to the IncP-7 incompatibility group that play important roles in the catabolism of several xenobiotics. pWW53 carried two distinct transposase-resolvase gene clusters (tnpAR modules), five short terminal inverted repeats (IRs), and three site-specific resolution (res) sites that are all typical of class II transposons. This organization of pWW53 suggested the four possible transposable regions, Tn4657 to Tn4660. The largest 86 kb region (Tn4657) spanned the three other regions, and Tn4657 and Tn4660 (62 kb) covered all of the 36 xyl genes for toluene catabolism. Our subsequent transposition experiments clarified that the three transposons, Tn4657 to Tn4659, indeed exhibit their transposability, and that pWW53 also generated another 37 kb toluene-catabolic transposon, Tn4656, which carried the two separated and inversely oriented segments of pWW53: the tnpRA-IR module of Tn4658 and a part of xyl gene clusters on Tn4657. The Tn4658 transposase was able to mediate the transposition of Tn4658, Tn4657, and Tn4656, while the Tn4659 transposase catalyzed only the transposition of Tn4659. Tn4656 was formed by the Tn4658 resolvase-mediated site-specific inversion between the two inversely oriented res sites on pWW53. These findings and comparison with other catabolic plasmids clearly indicate multiple copies of transposition-related genes and sites on one plasmid and their recombination activities contribute greatly to the diversification of plasmid structures as well as wide dissemination of the evolutionary common gene clusters in various plasmids.
ESTHER : Yano_2007_J.Mol.Biol_369_11
PubMedSearch : Yano_2007_J.Mol.Biol_369_11
PubMedID: 17408691

Title : Crystallization and preliminary crystallographic analysis of a haloalkane dehalogenase, DbjA, from Bradyrhizobium japonicum USDA110 - Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
Author(s) : Sato Y , Natsume R , Tsuda M , Damborsky J , Nagata Y , Senda T
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :294 , 2007
Abstract : Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. The haloalkane dehalogenase DbjA constitutes a novel substrate-specificity class with high catalytic activity for beta-methylated haloalkanes. In order to reveal the mechanism of its substrate specificity, DbjA has been crystallized using the hanging-drop vapour-diffusion method. The best crystals were obtained using the microseeding technique with a reservoir solution consisting of 17-19.5%(w/v) PEG 4000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 7.7-8.0. The space group of the DbjA crystal is P2(1)2(1)2, with unit-cell parameters a = 212.9, b = 117.8, c = 55.8 A. The crystal diffracts to 1.75 A resolution.
ESTHER : Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
PubMedSearch : Sato_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_294
PubMedID: 17401198

Title : Identification of a response regulator gene for catabolite control from a PCB-degrading beta-proteobacteria, Acidovorax sp. KKS102 - Ohtsubo_2006_Mol.Microbiol_60_1563
Author(s) : Ohtsubo Y , Goto H , Nagata Y , Kudo T , Tsuda M
Ref : Molecular Microbiology , 60 :1563 , 2006
Abstract : Acidovorax sp. (formally Pseudomonas sp.) strain KKS102 carries a bph operon for the degradation of PCB/biphenyl. Transcription from the pE promoter for the bph operon was found to be under catabolite control, i.e. the promoter activity was at a lower level when succinate, fumarate or acetate was added to the culture. Some mutations in the immediate upstream region of the pE promoter resulted in catabolite-insensitive and constitutively low promoter activity, suggesting that a transcriptional activator was involved in catabolite control. A genetic screen for a pE promoter activator identified two tandemly arranged genes, bphP and bphQ, that encoded proteins homologous to the sensor kinases and response regulators, respectively, of two-component regulatory system. In the bphPQ double mutant, pE promoter activity was weak and catabolite-insensitive, and a supply of the bphQ gene alone led to the restoration of the catabolite response. The mechanism of catabolite repression in KKS102 is explained in terms of inhibition of activation by BphQ. The genes highly similar to bphQ were found from several beta-proteobacteria, such as Burkholderia cenocepacia J2315, B. multivorans ATCC17616, B. xenovorans LB400 and Ralstonia solanacearum RS1085.
ESTHER : Ohtsubo_2006_Mol.Microbiol_60_1563
PubMedSearch : Ohtsubo_2006_Mol.Microbiol_60_1563
PubMedID: 16796688

Title : Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris - Nakamura_2006_Protein.Expr.Purif_46_85
Author(s) : Nakamura T , Zamocky M , Zdrahal Z , Chaloupkova R , Monincova M , Prokop Z , Nagata Y , Damborsky J
Ref : Protein Expr Purif , 46 :85 , 2006
Abstract : Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.
ESTHER : Nakamura_2006_Protein.Expr.Purif_46_85
PubMedSearch : Nakamura_2006_Protein.Expr.Purif_46_85
PubMedID: 16216524

Title : Complete nucleotide sequence of an exogenously isolated plasmid, pLB1, involved in gamma-hexachlorocyclohexane degradation - Miyazaki_2006_Appl.Environ.Microbiol_72_6923
Author(s) : Miyazaki R , Sato Y , Ito M , Ohtsubo Y , Nagata Y , Tsuda M
Ref : Applied Environmental Microbiology , 72 :6923 , 2006
Abstract : The alpha-proteobacterial strain Sphingobium japonicum UT26 utilizes a highly chlorinated pesticide, gamma-hexachlorocyclohexane (gamma-HCH), as a sole source of carbon and energy, and haloalkane dehalogenase LinB catalyzes the second step of gamma-HCH degradation in UT26. Functional complementation of a linB mutant of UT26, UT26DB, was performed by the exogenous plasmid isolation technique using HCH-contaminated soil, leading to our successful identification of a plasmid, pLB1, carrying the linB gene. Complete sequencing analysis of pLB1, with a size of 65,998 bp, revealed that it carries (i) 50 totally annotated coding sequences, (ii) an IS6100 composite transposon containing two copies of linB, and (iii) potential genes for replication, maintenance, and conjugative transfer with low levels of similarity to other homologues. A minireplicon assay demonstrated that a 2-kb region containing the predicted repA gene and its upstream region of pLB1 functions as an autonomously replicating unit in UT26. Furthermore, pLB1 was conjugally transferred from UT26DB to other alpha-proteobacterial strains but not to any of the beta- or gamma-proteobacterial strains examined to date. These results suggest that this exogenously isolated novel plasmid contributes to the dissemination of at least some genes for gamma-HCH degradation in the natural environment. To the best of our knowledge, this is the first detailed report of a plasmid involved in gamma-HCH degradation.
ESTHER : Miyazaki_2006_Appl.Environ.Microbiol_72_6923
PubMedSearch : Miyazaki_2006_Appl.Environ.Microbiol_72_6923
PubMedID: 16963556
Gene_locus related to this paper: sphpi-linb

Title : Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities - Sato_2005_Appl.Environ.Microbiol_71_4372
Author(s) : Sato Y , Monincova M , Chaloupkova R , Prokop Z , Ohtsubo Y , Minamisawa K , Tsuda M , Damborsky J , Nagata Y
Ref : Applied Environmental Microbiology , 71 :4372 , 2005
Abstract : Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.
ESTHER : Sato_2005_Appl.Environ.Microbiol_71_4372
PubMedSearch : Sato_2005_Appl.Environ.Microbiol_71_4372
PubMedID: 16085827

Title : Quantitative analysis of substrate specificity of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 - Kmunicek_2005_Biochemistry_44_3390
Author(s) : Kmunicek J , Hynkova K , Jedlicka T , Nagata Y , Negri A , Gago F , Wade RC , Damborsky J
Ref : Biochemistry , 44 :3390 , 2005
Abstract : Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty-five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants K(m) and k(cat) were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structure-activity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment, and volume:surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends nonlinearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity. A model with three statistically significant principal components explained 95% of the experimental data variability. van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248, and His272, (ii) tunnel residues Pro144, Asp147, Leu177, and Ala247, and (iii) second-shell residues Pro39 and Phe273. The tunnel and the second-shell residues represent the best targets for modulating specificity since their replacement does not lead to loss of functionality by disruption of the active site architecture. The mechanism of molecular adaptation toward a different specificity is discussed on the basis of quantitative comparison of models derived for two protein family members.
ESTHER : Kmunicek_2005_Biochemistry_44_3390
PubMedSearch : Kmunicek_2005_Biochemistry_44_3390
PubMedID: 15736949
Gene_locus related to this paper: sphpi-linb

Title : Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases - Jesenska_2005_Appl.Environ.Microbiol_71_6736
Author(s) : Jesenska A , Pavlova M , Strouhal M , Chaloupkova R , Tesinska I , Monincova M , Prokop Z , Bartos M , Pavlik I , Rychlik I , Mobius P , Nagata Y , Damborsky J
Ref : Applied Environmental Microbiology , 71 :6736 , 2005
Abstract : Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.
ESTHER : Jesenska_2005_Appl.Environ.Microbiol_71_6736
PubMedSearch : Jesenska_2005_Appl.Environ.Microbiol_71_6736
PubMedID: 16269704
Gene_locus related to this paper: myctu-linb

Title : Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26 - Nagata_2005_Appl.Environ.Microbiol_71_2183
Author(s) : Nagata Y , Prokop Z , Sato Y , Jerabek P , Kumar A , Ohtsubo Y , Tsuda M , Damborsky J
Ref : Applied Environmental Microbiology , 71 :2183 , 2005
Abstract : Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.
ESTHER : Nagata_2005_Appl.Environ.Microbiol_71_2183
PubMedSearch : Nagata_2005_Appl.Environ.Microbiol_71_2183
PubMedID: 15812056

Title : Identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 - Endo_2005_J.Bacteriol_187_847
Author(s) : Endo R , Kamakura M , Miyauchi K , Fukuda M , Ohtsubo Y , Tsuda M , Nagata Y
Ref : Journal of Bacteriology , 187 :847 , 2005
Abstract : Sphingomonas paucimobilis UT26 utilizes gamma-hexachlorocyclohexane (gamma-HCH) as a sole source of carbon and energy. In our previous study, we cloned and characterized genes that are involved in the conversion of gamma-HCH to maleylacetate (MA) via chlorohydroquinone (CHQ) in UT26. In this study, we identified and characterized an MA reductase gene, designated linF, that is essential for the utilization of gamma-HCH in UT26. A gene named linEb, whose deduced product showed significant identity to LinE (53%), was located close to linF. LinE is a novel type of ring cleavage dioxygenase that catalyzes the conversion of CHQ to MA. LinEb expressed in Escherichia coli transformed CHQ and 2,6-dichlorohydroquinone to MA and 2-chloromaleylacetate, respectively. Our previous and present results indicate that UT26 (i) has two gene clusters for degradation of chlorinated aromatic compounds via hydroquinone-type intermediates and (ii) uses at least parts of both clusters for gamma-HCH utilization.
ESTHER : Endo_2005_J.Bacteriol_187_847
PubMedSearch : Endo_2005_J.Bacteriol_187_847
PubMedID: 15659662
Gene_locus related to this paper: psepa-q6vqy9

Title : Crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 at 0.95 A resolution: dynamics of catalytic residues - Oakley_2004_Biochemistry_43_870
Author(s) : Oakley AJ , Klvana M , Otyepka M , Nagata Y , Wilce MC , Damborsky J
Ref : Biochemistry , 43 :870 , 2004
Abstract : We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 A resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C(alpha)-C(beta)-C(gamma) angle (126 degrees ) that deviated by 13.4 degrees from the "ideal" bond angle of 112.6 degrees. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C(alpha)-C(beta)-C(gamma) angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N(delta)(1) and Asp108 in conformation A gave the most exothermic reaction (DeltaH = -22 kcal/mol). With His272 doubly protonated at N(delta)(1) and N(epsilon)(2), the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C(alpha)-C(beta)-C(gamma) angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.
ESTHER : Oakley_2004_Biochemistry_43_870
PubMedSearch : Oakley_2004_Biochemistry_43_870
PubMedID: 14744129
Gene_locus related to this paper: sphpi-linb

Title : Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel - Chaloupkova_2003_J.Biol.Chem_278_52622
Author(s) : Chaloupkova R , Sykorova J , Prokop Z , Jesenska A , Monincova M , Pavlova M , Tsuda M , Nagata Y , Damborsky J
Ref : Journal of Biological Chemistry , 278 :52622 , 2003
Abstract : Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.
ESTHER : Chaloupkova_2003_J.Biol.Chem_278_52622
PubMedSearch : Chaloupkova_2003_J.Biol.Chem_278_52622
PubMedID: 14525993

Title : Catalytic mechanism of the maloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 - Prokop_2003_J.Biol.Chem_278_45094
Author(s) : Prokop Z , Monincova M , Chaloupkova R , Klvana M , Nagata Y , Janssen DB , Damborsky J
Ref : Journal of Biological Chemistry , 278 :45094 , 2003
Abstract : Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.
ESTHER : Prokop_2003_J.Biol.Chem_278_45094
PubMedSearch : Prokop_2003_J.Biol.Chem_278_45094
PubMedID: 12952988
Gene_locus related to this paper: sphpi-linb

Title : Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates - Streltsov_2003_Biochemistry_42_10104
Author(s) : Streltsov VA , Prokop Z , Damborsky J , Nagata Y , Oakley A , Wilce MC
Ref : Biochemistry , 42 :10104 , 2003
Abstract : The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.
ESTHER : Streltsov_2003_Biochemistry_42_10104
PubMedSearch : Streltsov_2003_Biochemistry_42_10104
PubMedID: 12939138
Gene_locus related to this paper: sphpi-linb

Title : Reconstruction of mycobacterial dehalogenase Rv2579 by cumulative mutagenesis of haloalkane dehalogenase LinB - Nagata_2003_Appl.Environ.Microbiol_69_2349
Author(s) : Nagata Y , Prokop Z , Marvanova S , Sykorova J , Monincova M , Tsuda M , Damborsky J
Ref : Applied Environmental Microbiology , 69 :2349 , 2003
Abstract : The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.
ESTHER : Nagata_2003_Appl.Environ.Microbiol_69_2349
PubMedSearch : Nagata_2003_Appl.Environ.Microbiol_69_2349
PubMedID: 12676719

Title : Halide-stabilizing residues of haloalkane dehalogenases studied by quantum mechanic calculations and site-directed mutagenesis - Bohac_2002_Biochemistry_41_14272
Author(s) : Bohac M , Nagata Y , Prokop Z , Prokop M , Monincova M , Tsuda M , Koca J , Damborsky J
Ref : Biochemistry , 41 :14272 , 2002
Abstract : Haloalkane dehalogenases catalyze cleavage of the carbon-halogen bond in halogenated aliphatic compounds, resulting in the formation of an alcohol, a halide, and a proton as the reaction products. Three structural features of haloalkane dehalogenases are essential for their catalytic performance: (i) a catalytic triad, (ii) an oxyanion hole, and (iii) the halide-stabilizing residues. Halide-stabilizing residues are not structurally conserved among different haloalkane dehalogenases. The level of stabilization of the transition state structure of S(N)2 reaction and halide ion provided by each of the active site residues in the enzymes DhlA, LinB, and DhaA was quantified by quantum mechanic calculations. The residues that significantly stabilize the halide ion were assigned as the primary (essential) or the secondary (less important) halide-stabilizing residues. Site-directed mutagenesis was conducted with LinB enzyme to confirm location of its primary halide-stabilizing residues. Asn38Asp, Asn38Glu, Asn38Phe, Asn38Gln, Trp109Leu, Phe151Leu, Phe151Trp, Phe151Tyr, and Phe169Leu mutants of LinB were constructed, purified, and kinetically characterized. The following active site residues were classified as the primary halide-stabilizing residues: Trp125 and Trp175 of DhlA; Asn38 and Trp109 of LinB; and Asn41 and Trp107 of DhaA. All these residues make a hydrogen bond with the halide ion released from the substrate molecule, and their substitution results in enzymes with significantly modified catalytic properties. The following active site residues were classified as the secondary halide-stabilizing residues: Phe172, Pro223, and Val226 of DhlA; Trp207, Pro208, and Ile211 of LinB; and Phe205, Pro206, and Ile209 of DhaA. The differences in the halide stabilizing residues of three haloalkane dehalogenases are discussed in the light of molecular adaptation of these enzymes to their substrates.
ESTHER : Bohac_2002_Biochemistry_41_14272
PubMedSearch : Bohac_2002_Biochemistry_41_14272
PubMedID: 12450392
Gene_locus related to this paper: sphpi-linb

Title : Exploring the structure and activity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26: evidence for product- and water-mediated inhibition - Oakley_2002_Biochemistry_41_4847
Author(s) : Oakley AJ , Prokop Z , Bohac M , Kmunicek J , Jedlicka T , Monincova M , Kuta-Smatanova I , Nagata Y , Damborsky J , Wilce MC
Ref : Biochemistry , 41 :4847 , 2002
Abstract : The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for example, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.
ESTHER : Oakley_2002_Biochemistry_41_4847
PubMedSearch : Oakley_2002_Biochemistry_41_4847
PubMedID: 11939779
Gene_locus related to this paper: sphpi-linb

Title : Structure-specificity relationships for haloalkane dehalogenases - Damborsky_2001_Environ.Toxicol.Chem_20_2681
Author(s) : Damborsky J , Rorije E , Jesenska A , Nagata Y , Klopman G , Peijnenburg WJ
Ref : Environ Toxicol Chem , 20 :2681 , 2001
Abstract : A structural analysis of the substrate specificity of hydrolytic dehalogenases originating from three different bacterial isolates has been performed using the multiple computer-automated structure evaluation methodology. This methodology identifies structural fragments in substrate molecules that either activate or deactivate biological processes. The analysis presented in this contribution is based on newly measured dehalogenation data combined with data from the literature (91 substrates). The enzymes under study represent different specificity classes of haloalkane dehalogenases (haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, Rhodococcus erythropolis Y2, and Sphingomonas paucimobilis UT26). Three sets of structural rules have been identified to explain their substrate specificity and to predict activity for untested substrates. Predictions of activity and inactivity based on the structural rules from this analysis were provided for those compounds that were not yet tested experimentally. Predictions were also made for the compounds with available experimental data not used for the model construction (i.e., the external validation set). Correct predictions were obtained for 28 of 30 compounds in the validation set. Incorrect predictions were noted for two substrates outside the chemical domain of the set of compounds for which the structural rules were generated. A mechanistic interpretation of the structural rules generated provided a fundamental understanding of the structure-specificity relationships for the family of haloalkane dehalogenases.
ESTHER : Damborsky_2001_Environ.Toxicol.Chem_20_2681
PubMedSearch : Damborsky_2001_Environ.Toxicol.Chem_20_2681
PubMedID: 11764149

Title : Biochemical characterization of broad-specificity enzymes using multivariate experimental design and a colorimetric microplate assay: characterization of the haloalkane dehalogenase mutants - Marvanova_2001_J.Microbiol.Methods_44_149
Author(s) : Marvanova S , Nagata Y , Wimmerova M , Sykorova J , Hynkova K , Damborsky J
Ref : J Microbiol Methods , 44 :149 , 2001
Abstract : The pH indicator dye-based colorimetric method and multivariate experimental design were used for the systematic biochemical characterization of the broad-specificity enzymes haloalkane dehalogenases. Halogenated compounds for characterization of the enzymes were selected using Principal Component Analysis. The substrates were characterised by 24 physico-chemical and structural descriptors. Thirty-four substrates were selected for testing out of 194 halogenated compounds. Relative activities determined using the optimised colorimetric microplate assay were validated against the catalytic constants determined by gas chromatography. The applicability of the assay was tested with F151L, F154L and F169L mutants of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26.
ESTHER : Marvanova_2001_J.Microbiol.Methods_44_149
PubMedSearch : Marvanova_2001_J.Microbiol.Methods_44_149
PubMedID: 11165344

Title : Expression of the bph genes involved in biphenyl\/PCB degradation in Pseudomonas sp. KKS102 induced by the biphenyl degradation intermediate, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid - Ohtsubo_2000_Gene_256_223
Author(s) : Ohtsubo Y , Nagata Y , Kimbara K , Takagi M , Ohta A
Ref : Gene , 256 :223 , 2000
Abstract : The bph genes involved in PCB/biphenyl degradation in Pseudomonas sp. KKS102 are clustered as bphEGFA1A2A3BCDA4R. The bph genes are inducibly expressed in the presence of biphenyl. In order to understand the induction more fully, the inducer of bph gene expression was investigated. To identify the inducer molecule, we constructed four deletion mutants of the structural genes and analyzed the inducibility of the bphE gene in each mutant strain. In the wild-type cell and the bphD deletion mutant, the levels of the bphE transcript were enhanced in the presence of biphenyl. On the other hand, in the bphA, bphB, and bphC deletion mutants, levels of the bphE transcript were not enhanced in the presence of biphenyl. These results demonstrated that the series of reactions catalyzed by biphenyl dioxygenase (BphA), dihydrodiol dehydrogenase (BphB), and 2, 3-dihydroxybiphenyl dioxygenase (BphC) are necessary to convert biphenyl to the inducer. It is known that these reactions convert biphenyl to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), and it was found that the expression of the bph genes was induced by purified HOPDA. These results clearly indicate that HOPDA is the inducer of the bph genes in KKS102.
ESTHER : Ohtsubo_2000_Gene_256_223
PubMedSearch : Ohtsubo_2000_Gene_256_223
PubMedID: 11054551

Title : Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 - Marek_2000_Biochemistry_39_14082
Author(s) : Marek J , Vevodova J , Smatanova IK , Nagata Y , Svensson LA , Newman J , Takagi M , Damborsky J
Ref : Biochemistry , 39 :14082 , 2000
Abstract : The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.
ESTHER : Marek_2000_Biochemistry_39_14082
PubMedSearch : Marek_2000_Biochemistry_39_14082
PubMedID: 11087355
Gene_locus related to this paper: sphpi-linb

Title : Determination of haloalkane dehalogenase activity by capillary zone electrophoresis - Glatz_2000_J.Chromatogr.A_895_219
Author(s) : Glatz Z , Marini MV , Wimmerova M , Damborsky J , Nagata Y
Ref : Journal of Chromatography A , 895 :219 , 2000
Abstract : A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 microm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25 degrees C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.
ESTHER : Glatz_2000_J.Chromatogr.A_895_219
PubMedSearch : Glatz_2000_J.Chromatogr.A_895_219
PubMedID: 11105865

Title : Two different types of dehalogenases, LinA and LinB, involved in gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26 are localized in the periplasmic space without molecular processing - Nagata_1999_J.Bacteriol_181_5409
Author(s) : Nagata Y , Futamura A , Miyauchi K , Takagi M
Ref : Journal of Bacteriology , 181 :5409 , 1999
Abstract : gamma-Hexachlorocyclohexane (gamma-HCH) is one of several highly chlorinated insecticides that cause serious environmental problems. The cellular proteins of a gamma-HCH-degrading bacterium, Sphingomonas paucimobilis UT26, were fractionated into periplasmic, cytosolic, and membrane fractions after osmotic shock. Most of two different types of dehalogenase, LinA (gamma-hexachlorocyclohexane dehydrochlorinase) and LinB (1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase), that are involved in the early steps of gamma-HCH degradation in UT26 was detected in the periplasmic fraction and had not undertaken molecular processing. Furthermore, immunoelectron microscopy clearly showed that LinA and LinB are periplasmic proteins. LinA and LinB both lack a typical signal sequence for export, so they may be secreted into the periplasmic space via a hitherto unknown mechanism.
ESTHER : Nagata_1999_J.Bacteriol_181_5409
PubMedSearch : Nagata_1999_J.Bacteriol_181_5409
PubMedID: 10464214
Gene_locus related to this paper: sphpi-linb

Title : Identification of the catalytic triad in the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 - Hynkova_1999_FEBS.Lett_446_177
Author(s) : Hynkova K , Nagata Y , Takagi M , Damborsky J
Ref : FEBS Letters , 446 :177 , 1999
Abstract : The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the gamma-hexachlorocyclohexane degradation. This enzyme hydrolyses a broad range of halogenated aliphatic compounds via an alkyl-enzyme intermediate. LinB is believed to belong to the family of alpha/beta-hydrolases which employ a catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. The position of the catalytic triad within the sequence of LinB was probed by a site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be D108, H272 and E132. The topological location of the catalytic acid (E132) is after the beta-strand six which corresponds to the location of catalytic acid in the pancreatic lipase, but not in the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10 which contains the catalytic acid after the beta-strand seven.
ESTHER : Hynkova_1999_FEBS.Lett_446_177
PubMedSearch : Hynkova_1999_FEBS.Lett_446_177
PubMedID: 10100638
Gene_locus related to this paper: sphpi-linb

Title : Crystallization and preliminary X-ray diffraction analysis of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 - Smatanova_1999_Acta.Crystallogr.D.Biol.Crystallogr_55_1231
Author(s) : Smatanova I , Nagata Y , Svensson LA , Takagi M , Marek J
Ref : Acta Crystallographica D Biol Crystallogr , 55 :1231 , 1999
Abstract : Haloalkane hydrolytic dehalogenase LinB from Sphingomonas paucimobilis UT26, an enzyme which releases chloride or bromide anion from n-halogenated alkanes and has a broad range of substrate specificity, was crystallized using the hanging-drop vapour-diffusion method at 278 K. The best crystals were obtained by microseeding with a precipitant containing 18-20%(w/v) PEG 6000, 0.2 M calcium acetate and 0.1 M Tris-HCl pH 8.9. The crystals diffract to at least 1.60 A using synchrotron X-ray under cryogenic (100 K) conditions. They belong to the orthorhombic space group P21212 with unit-cell parameters a = 50.29, b = 71.70, c = 72.73 A. The asymmetric unit contains one molecule of the enzyme.
ESTHER : Smatanova_1999_Acta.Crystallogr.D.Biol.Crystallogr_55_1231
PubMedSearch : Smatanova_1999_Acta.Crystallogr.D.Biol.Crystallogr_55_1231
PubMedID: 10329794
Gene_locus related to this paper: sphpi-linb

Title : Construction and characterization of histidine-tagged haloalkane dehalogenase (LinB) of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26 - Nagata_1999_Protein.Expr.Purif_17_299
Author(s) : Nagata Y , Hynkova K , Damborsky J , Takagi M
Ref : Protein Expr Purif , 17 :299 , 1999
Abstract : The linB gene product (LinB), which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26, is a member of haloalkane dehalogenases with a broad range of substrate specificity. Elucidation of the factors determining its substrate specificity is of interest. Aiming to facilitate purification of recombinant LinB protein for site-directed mutagenesis analysis, a 6-histidyl tail was added to the C-terminus of LinB. The His-tagged LinB was specifically bound with Ni-NTA resin in the buffer containing 10 mM imidazole. After elution with 500 mM imidazole, quantitative recovery of protein occurred. The steady-state kinetic parameters of the His-tagged LinB for four substrates were in good agreement with that of wild-type recombinant LinB. Although the His-tagged LinB expressed in an average of 80% of the activity of the wild type LinB for 10 different substrates, the decrease was very similar for different substrates with the standard deviation of 5.5%. The small activity reduction is independent of the substrate shape, size, or number of substituents, indicating that the His-tagged LinB can be used for further mutagenesis studies. To confirm the suitability of this system for mutagenesis studies, two mutant proteins with substitution in putative halide binding residues (W109 and F151) were constructed, purified, and tested for activity. As expected, complete loss in activity of W109L and sustained activity of F151W were observed.
ESTHER : Nagata_1999_Protein.Expr.Purif_17_299
PubMedSearch : Nagata_1999_Protein.Expr.Purif_17_299
PubMedID: 10545279

Title : Purification and characterization of a haloalkane dehalogenase of a new substrate class from a gamma-hexachlorocyclohexane-degrading bacterium, Sphingomonas paucimobilis UT26 - Nagata_1997_Appl.Environ.Microbiol_63_3707
Author(s) : Nagata Y , Miyauchi K , Damborsky J , Manova K , Ansorgova A , Takagi M
Ref : Applied Environmental Microbiology , 63 :3707 , 1997
Abstract : The linB gene product (LinB), 1,3,4,6-tetrachloro-1,4-cyclohexadiene halidohydrolase, which is involved in the degradation of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26 (Y. Nagata, T. Nariya, R. Ohtomo, M. Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 175:6403-6410, 1993), was overproduced in E. coli and purified to homogeneity. The molecular mass of LinB was deduced to be 30 kDa by gel filtration chromatography and 32 kDa by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, indicating that LiuB is a monomeric enzyme. The optimal pH for activity was 8.2. Not only monochloroalkanes (C3 to C10) but also dichloroalkanes, bromoalkanes, and chlorinated allphatic alcohols were good substrates for LinB, suggesting that LinB shares properties with another haloalkane dehalogenase, DhlA (S. Keuning, D.B. Janssen, and B. Witholt, J. Bacteriol. 163:635-639, 1985), which shows significant similarity to LinB in primary structure (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989) but not in substrate specificity. Principal component analysis of substrate activities of various haloalkane dehalogenases suggested that LinB probably constitutes a new substrate specificity class within this group of enzymes.
ESTHER : Nagata_1997_Appl.Environ.Microbiol_63_3707
PubMedSearch : Nagata_1997_Appl.Environ.Microbiol_63_3707
PubMedID: 9293022
Gene_locus related to this paper: sphpi-linb

Title : Cloning and sequencing of a dehalogenase gene encoding an enzyme with hydrolase activity involved in the degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis - Nagata_1993_J.Bacteriol_175_6403
Author(s) : Nagata Y , Nariya T , Ohtomo R , Fukuda M , Yano K , Takagi M
Ref : Journal of Bacteriology , 175 :6403 , 1993
Abstract : In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is converted by two steps of dehydrochlorination to a chemically unstable intermediate, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), which is then metabolized to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by two steps of hydrolytic dehalogenation via the chemically unstable intermediate 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding the enzyme responsible for the conversion of the chemically unstable intermediates 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida PpY101LA into which the linA gene had been introduced by Tn5. An 8-kb BglII fragment from one of the cosmid clones, which could convert gamma-HCH to 2,5-DDOL, was subcloned, and subsequent deletion analyses revealed that a ca. 1.1-kb region was responsible for the activity. Nucleotide sequence analysis revealed an open reading frame (designated the linB gene) of 885 bp within the region. The deduced amino acid sequence of LinB showed significant similarity to hydrolytic dehalogenase, DhlA (D. B. Janssen, F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J. Bacteriol. 171:6791-6799, 1989). The protein product of the linB gene was 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only 1-chlorobutane but also 1-chlorodecane (C10) and 2-chlorobutane, which are poor substrates for other dehalogenases, were good substrates for LinB, suggesting that LinB may be a member of haloalkane dehalogenases with broad-range specificity for substrates.
ESTHER : Nagata_1993_J.Bacteriol_175_6403
PubMedSearch : Nagata_1993_J.Bacteriol_175_6403
PubMedID: 7691794
Gene_locus related to this paper: sphpi-linb