Kanaya S

References (24)

Title : Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain - Murata_2017_Biochemistry_56_6281
Author(s) : Murata D , Okano H , Angkawidjaja C , Akutsu M , Tanaka SI , Kitahara K , Yoshizawa T , Matsumura H , Kado Y , Mizohata E , Inoue T , Sano S , Koga Y , Kanaya S , Takano K
Ref : Biochemistry , 56 :6281 , 2017
Abstract : Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long alpha-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long alpha-helical domain has three alpha-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.
ESTHER : Murata_2017_Biochemistry_56_6281
PubMedSearch : Murata_2017_Biochemistry_56_6281
PubMedID: 29094929

Title : Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension - Okano_2015_Protein.Sci_24_93
Author(s) : Okano H , Hong X , Kanaya E , Angkawidjaja C , Kanaya S
Ref : Protein Science , 24 :93 , 2015
Abstract : The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1 - 6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome-derived enzymes, LC-Est1 is characterized by the presence of a long N-terminal extension (LNTE, residues 26-283) between a putative signal peptide (residues 1-25) and a C-terminal esterase domain (residues 284-510). A putative esterase from Candidatus Solibacter usitatus (CSu-Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC-Est1. To examine whether LC-Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC-Est1 (residues 26-510), LC-Est1C (residues 284-510), and LC-Est1C* (residues 304-510) were overproduced in E. coli, purified, and characterized. LC-Est1C* was only used for structural analysis. The crystal structure of LC-Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC-Est1C was lower than that of LC-Est1 by 60%, although its substrate specificity was similar to that of LC-Est1. LC-Est1C was less stable than LC-Est1 by 3.3 degrees C. These results suggest that LNTE of LC-Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.
ESTHER : Okano_2015_Protein.Sci_24_93
PubMedSearch : Okano_2015_Protein.Sci_24_93
PubMedID: 25348365
Gene_locus related to this paper: 9bact-3WYDseq , 9bact-a0a097i5b4

Title : Calcium-independent opening of lid1 of a family I.3 lipase by a single Asp to Arg mutation at the calcium-binding site - Cheng_2014_Protein.Eng.Des.Sel_27_169
Author(s) : Cheng M , Angkawidjaja C , Koga Y , Kanaya S
Ref : Protein Engineering Des Sel , 27 :169 , 2014
Abstract : A family I.3 lipase from Pseudomonas sp. MIS38 (PML) has two lids, lid1 and lid2, which are open when it exhibits activity. A single calcium ion is required to anchor lid1 in the open conformation by coordination with two acidic residues (Asp153 and Asp157) in lid1 and three other residues. Lid1 adopts a long alpha-helix in the open conformation, whereas it is sharply bent within this helix, such that Asp153 and Asp157 are distantly located to each other, in the closed conformation. To examine whether the mutation of Asp153 or Asp157 to a positively charged residue allows two residues at Positions 153 and 157 to come close with each other and thereby stabilizes the open conformation of lid1 even in the absence of calcium ions, five single mutant proteins (D153K-, D153R-, D153A-, D157K- and D157R-PMLs) and two double mutant proteins (D153A/D157A- and D153R/D157N-PMLs) were constructed. Of these mutant proteins, only D153R-PML exhibited activity in the absence of calcium ions. Its lipase and esterase activities were 7-fold lower and 4-fold higher than those of PML, respectively. These activities were lost by the mutation of Asp157 to Asn. These results suggest that lid1 of D153R-PML opens even in the absence of calcium ions due to electrostatic attraction between Arg153 and Asp157.
ESTHER : Cheng_2014_Protein.Eng.Des.Sel_27_169
PubMedSearch : Cheng_2014_Protein.Eng.Des.Sel_27_169
PubMedID: 24737906

Title : Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase - Sulaiman_2014_Biochemistry_53_1858
Author(s) : Sulaiman S , You DJ , Kanaya E , Koga Y , Kanaya S
Ref : Biochemistry , 53 :1858 , 2014
Abstract : The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 A resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 degrees C were 86.2 degrees C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, DeltaG(H2O), was 41.8 kJ mol(-1) at 30 degrees C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 x 10(-6) s(-1) at 50 degrees C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 degrees C) was considerably lower than the T1/2 value. It increased by 10 degrees C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 degrees C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.
ESTHER : Sulaiman_2014_Biochemistry_53_1858
PubMedSearch : Sulaiman_2014_Biochemistry_53_1858
PubMedID: 24593046
Gene_locus related to this paper: 9bact-g9by57

Title : Crystallization and X-ray structure determination of a thermoalkalophilic lipase from Geobacillus SBS-4S - Tayyab_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_355
Author(s) : Tayyab M , Rashid N , Angkawidjaja C , Kanaya S , Akhtar M
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 69 :355 , 2013
Abstract : A thermoalkalophilic lipase (LIPSBS) from the newly isolated Geobacillus strain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBS using the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 55.13, b = 71.75, c = 126.26A. The structure was determined at 1.6A resolution by the molecular-replacement method using the lipase from G. stearothermophilus L1 as a model.
ESTHER : Tayyab_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_355
PubMedSearch : Tayyab_2013_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_69_355
PubMedID: 23545637
Gene_locus related to this paper: bacsp-lip

Title : Enhancement of the Enzymatic Activity of Escherichia coli Acetyl Esterase by a Double Mutation Obtained by Random Mutagenesis - Kobayashi_2012_Biosci.Biotechnol.Biochem_76_2082
Author(s) : Kobayashi R , Hirano N , Kanaya S , Haruki M
Ref : Biosci Biotechnol Biochem , 76 :2082 , 2012
Abstract : A double mutant of Escherichia coli acetyl esterase (EcAE) with enhanced enzymatic activity was obtained by random mutagenesis using error-prone PCR and screening for enzymatic activity by observing halo formation on a tributyrin plate. The mutant contained Leu97Phe (L97F) and Leu209Phe (L209F) mutations. Single mutants L97F and L209F were also constructed and analyzed for kinetic parameters, as well as double mutant L97F/L209F. Kinetic analysis using p-nitrophenyl butyrate as substrate indicated that the k(cat) values of L97F and L97F/L209F were larger than that of the wild-type enzyme, by 8.3-fold and 12-fold respectively, whereas no significant change was observed in the k(cat) value of L209F. The K(m) values of L209F and L97F/L209F were smaller than that of the wild-type enzyme, by 2.9-fold and 2.4-fold respectively, whereas no significant change was observed in the K(m) value of L97F. These results indicate that a combination of an increase in k(cat) values due to the L97F mutation and a decrease in K(m) value due to the L209F mutation renders the k(cat)/K(m) value of the double mutant enzyme 29-fold higher than that of the wild-type enzyme.
ESTHER : Kobayashi_2012_Biosci.Biotechnol.Biochem_76_2082
PubMedSearch : Kobayashi_2012_Biosci.Biotechnol.Biochem_76_2082
PubMedID: 23132590

Title : Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures - Cheng_2012_FEBS.J_279_3727
Author(s) : Cheng M , Angkawidjaja C , Koga Y , Kanaya S
Ref : Febs J , 279 :3727 , 2012
Abstract : A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (DeltaL2-PML) was constructed by deleting residues 35-64 of PML. DeltaL2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of DeltaL2-PML did not. These results indicate that PML undergoes interfacial activation, while DeltaL2-PML does not. The activities of DeltaL2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of DeltaL2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML.
ESTHER : Cheng_2012_FEBS.J_279_3727
PubMedSearch : Cheng_2012_FEBS.J_279_3727
PubMedID: 22863357
Gene_locus related to this paper: psesp-Q9RBY1

Title : Structure and stability of a thermostable carboxylesterase from the thermoacidophilic archaeon Sulfolobus tokodaii. - Angkawidjaja_2012_Febs.J_279_3071
Author(s) : Angkawidjaja C , Koga Y , Takano K , Kanaya S
Ref : Febs J , 279 :3071 , 2012
Abstract : Hormone sensitive lipase (HSL) family is a family of carboxylesterases and lipases with similarity to mammalian HSL. Thermophilic enzymes of this family have high potential for the use in biocatalysis. We prepared and crystallized a carboxylesterase of HSL family from Sulfolobus tokodaii (Sto-Est), and determined its structures in the presence and absence of an inhibitor. Sto-Est forms a dimer in solution and the crystal structure suggests the presence of a stable biological dimer. We identified a residue close to the dimer interface, R267, which is conserved in the archaeal enzymes of HSL family and is in close proximity with the same residue from the other monomer. Mutations of R267 to Glu, Gly and Lys were conducted and the resultant R267 mutants were characterized and crystallized. The structures of R267E, R267G and R267K are highly similar to that of Sto-Est with only slight differences in atomic coordinates. The dimerized state of R267E and R267G are unstable under denaturing condition or at high temperature, as shown by urea-induced dimer dissociation experiment and MD simulation. R267E is the most unstable mutant protein, followed by R267G and R267K, as shown by the thermal denaturation curve and optimum temperature for activity. From the data we discuss the importance of R267 in maintaining the dimer integrity of Sto-Est.
ESTHER : Angkawidjaja_2012_Febs.J_279_3071
PubMedSearch : Angkawidjaja_2012_Febs.J_279_3071
PubMedID: 22748144
Gene_locus related to this paper: sulto-ST0071

Title : Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach - Sulaiman_2012_Appl.Environ.Microbiol_78_1556
Author(s) : Sulaiman S , Yamato S , Kanaya E , Kim JJ , Koga Y , Takano K , Kanaya S
Ref : Applied Environmental Microbiology , 78 :1556 , 2012
Abstract : The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50 degrees C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70 degrees C and 7 min at 80 degrees C. LC-cutinase* had an ability to degrade poly(epsilon-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/mukat of pNP-butyrate-degrading activity) at pH 8.0 and 50 degrees C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.
ESTHER : Sulaiman_2012_Appl.Environ.Microbiol_78_1556
PubMedSearch : Sulaiman_2012_Appl.Environ.Microbiol_78_1556
PubMedID: 22194294
Gene_locus related to this paper: 9bact-g9by57

Title : Importance of an extreme C-terminal motif of a family I.3 lipase for stability - Kuwahara_2011_Protein.Eng.Des.Sel_24_411
Author(s) : Kuwahara K , Angkawidjaja C , Koga Y , Takano K , Kanaya S
Ref : Protein Engineering Des Sel , 24 :411 , 2011
Abstract : A five-residue sequence motif (VTLVG) located at positions 15-19 from the C-terminus of family I.3 lipase from Pseudomonas sp. MIS38 (PML) and an extreme C-terminal motif (DGIVIA) located at the C-terminus of PML are relatively well conserved in the passenger proteins of type 1 secretion system (T1SS). To analyze the role of these motifs, four mutant proteins of PML (PMLDelta5, PMLDelta10, 3A-PML and 2A-PML) were constructed. PMLDelta5 and PMLDelta10 lack the C-terminal 5 and 10 residues of PML, respectively. 3A-PML has triple mutations within an extreme C-terminal motif and 2A-PML has double mutations within a five-residue sequence motif. Secretion of these proteins was analyzed using Escherichia coli DH5 cells carrying Lip system (T1SS for family I.3 lipase). The secretion level of 2A-PML was dramatically reduced when compared with that of PML, whereas the secretion level of 3A-PML was comparable to that of PML, indicating that a five-residue sequence motif, instead of an extreme C-terminal motif, is required for secretion of PML. None of the mutations and truncations seriously affects the enzymatic activity of PML. However, 3A-PML, PMLDelta5 and PMLDelta10 were less stable than PML by 2.1, 7.6 and 7.6 degrees C in T(1/2), respectively, and by 5.0, 21.3 and 17.9 kJ/mol in DeltaG(H(2)O), respectively. These results indicate that an extreme C-terminal motif of PML is important for stability.
ESTHER : Kuwahara_2011_Protein.Eng.Des.Sel_24_411
PubMedSearch : Kuwahara_2011_Protein.Eng.Des.Sel_24_411
PubMedID: 21216728

Title : X-ray crystallographic and MD simulation studies on the mechanism of interfacial activation of a family I.3 lipase with two lids - Angkawidjaja_2010_J.Mol.Biol_400_82
Author(s) : Angkawidjaja C , Matsumura H , Koga Y , Takano K , Kanaya S
Ref : Journal of Molecular Biology , 400 :82 , 2010
Abstract : The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two alpha-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 A resolution in the presence of Ca(2+) and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca(2+)-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca(2+) and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.
ESTHER : Angkawidjaja_2010_J.Mol.Biol_400_82
PubMedSearch : Angkawidjaja_2010_J.Mol.Biol_400_82
PubMedID: 20438738
Gene_locus related to this paper: psesp-Q9RBY1

Title : Importance of the Ca2+-binding sites in the N-catalytic domain of a family I.3 lipase for activity and stability - Kuwahara_2008_Protein.Eng.Des.Sel_21_737
Author(s) : Kuwahara K , Angkawidjaja C , Matsumura H , Koga Y , Takano K , Kanaya S
Ref : Protein Engineering Des Sel , 21 :737 , 2008
Abstract : A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains three Ca(2+)-binding sites (Ca1-Ca3) in the N-catalytic domain. Of them, the Ca1 site is formed only in an open conformation. To analyze the role of these Ca(2+)-binding sites, three mutant proteins D157A-PML, D275A-PML and D337A-PML, which are designed to remove the Ca1, Ca2 and Ca3 sites, respectively, were constructed. Of them, the crystal structures of D157A-PML and D337A-PML in a closed conformation were determined. Both structures are nearly identical to that of the wild-type protein, except that the Ca3 site is missing in the D337A-PML structure. D157A-PML was as stable as the wild-type protein. Nevertheless, it exhibited little lipase and very weak esterase activities. D275A-PML was less stable than the wild-type protein by approximately 5 degrees C in T(1/2). It exhibited weak but significant lipase and esterase activities when compared with the wild-type protein. D337A-PML was also less stable than the wild-type protein by approximately 5 degrees C in T(1/2) but was fully active. These results suggest that the Ca1 site is required to make the active site fully open by anchoring lid 1. The Ca2 and Ca3 sites contribute to the stabilization of PML. The Ca2 site is also required to make PML fully active.
ESTHER : Kuwahara_2008_Protein.Eng.Des.Sel_21_737
PubMedSearch : Kuwahara_2008_Protein.Eng.Des.Sel_21_737
PubMedID: 18987131
Gene_locus related to this paper: psesp-Q9RBY1

Title : Crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation - Angkawidjaja_2007_FEBS.Lett_581_5060
Author(s) : Angkawidjaja C , You DJ , Matsumura H , Kuwahara K , Koga Y , Takano K , Kanaya S
Ref : FEBS Letters , 581 :5060 , 2007
Abstract : The crystal structure of a family I.3 lipase from Pseudomonas sp. MIS38 in a closed conformation was determined at 1.5A resolution. This structure highly resembles that of Serratia marcescens LipA in an open conformation, except for the structures of two lids. Lid1 is anchored by a Ca2+ ion (Ca1) in an open conformation, but lacks this Ca1 site and greatly changes its structure and position in a closed conformation. Lid2 forms a helical hairpin in an open conformation, but does not form it and covers the active site in a closed conformation. Based on these results, we discuss on the lid-opening mechanism.
ESTHER : Angkawidjaja_2007_FEBS.Lett_581_5060
PubMedSearch : Angkawidjaja_2007_FEBS.Lett_581_5060
PubMedID: 17923123
Gene_locus related to this paper: psesp-Q9RBY1

Title : Extracellular overproduction and preliminary crystallographic analysis of a family I.3 lipase - Angkawidjaja_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_187
Author(s) : Angkawidjaja C , You DJ , Matsumura H , Koga Y , Takano K , Kanaya S
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :187 , 2007
Abstract : A family I.3 lipase from Pseudomonas sp. MIS38 was secreted from Escherichia coli cells to the external medium, purified and crystallized and preliminary crystallographic studies were performed. The crystal was grown at 277 K by the hanging-drop vapour-diffusion method. Native X-ray diffraction data were collected to 1.7 A resolution using synchrotron radiation at station BL38B1, SPring-8. The crystal belongs to space group P2(1), with unit-cell parameters a = 48.79, b = 84.06, c = 87.04 A. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.73 A3 Da(-1) and the solvent content was 55%.
ESTHER : Angkawidjaja_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_187
PubMedSearch : Angkawidjaja_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_187
PubMedID: 17329810
Gene_locus related to this paper: psesp-Q9RBY1

Title : Comparative analysis of argK-tox clusters and their flanking regions in phaseolotoxin-producing Pseudomonas syringae pathovars - Genka_2006_J.Mol.Evol_63_401
Author(s) : Genka H , Baba T , Tsuda M , Kanaya S , Mori H , Yoshida T , Noguchi MT , Tsuchiya K , Sawada H
Ref : Journal of Molecular Evolution , 63 :401 , 2006
Abstract : DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181-10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064-11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named "tox islands." The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437-457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands.
ESTHER : Genka_2006_J.Mol.Evol_63_401
PubMedSearch : Genka_2006_J.Mol.Evol_63_401
PubMedID: 16927007
Gene_locus related to this paper: psesy-PSPTO4540

Title : Importance of a repetitive nine-residue sequence motif for intracellular stability and functional structure of a family I.3 lipase - Angkawidjaja_2005_FEBS.Lett_579_4707
Author(s) : Angkawidjaja C , Paul A , Koga Y , Takano K , Kanaya S
Ref : FEBS Letters , 579 :4707 , 2005
Abstract : PML5 is a functional derivative of a family I.3 lipase from Pseudomonas sp. MIS38 and contains five repeats of a nine-residue sequence motif. Two aspartate residues within the second and third repetitive sequences of PML5 were replaced by Ala. The secretion level, intracellular accumulation level, and stability of the resultant mutant protein were greatly reduced as compared to those of PML5. In addition, this mutant protein was inactive and did not bind Ca2+ ion. We propose that the repetitive sequences of PML5 form a beta-roll structure in the cells and thereby contribute to the intracellular stability and secretion efficiency of the protein.
ESTHER : Angkawidjaja_2005_FEBS.Lett_579_4707
PubMedSearch : Angkawidjaja_2005_FEBS.Lett_579_4707
PubMedID: 16098975

Title : Cloning and characterization of the gene cluster encoding arthrofactin synthetase from Pseudomonas sp. MIS38 - Roongsawang_2003_Chem.Biol_10_869
Author(s) : Roongsawang N , Hase K , Haruki M , Imanaka T , Morikawa M , Kanaya S
Ref : Chemical Biology , 10 :869 , 2003
Abstract : Arthrofactin is a potent cyclic lipopeptide-type biosurfactant produced by Pseudomonas sp. MIS38. In this work, an arthrofactin synthetase gene cluster (arf) spanning 38.7 kb was cloned and characterized. Three genes termed arfA, arfB, and arfC encode ArfA, ArfB, and ArfC, containing two, four, and five functional modules, respectively. Each module bears condensation, adenylation, and thiolation domains, like other nonribosomal peptide synthetases. However, unlike most of them, none of the 11 modules possess the epimerization domain responsible for the conversion of amino acid residues from L to D form. Possible L- and D-Leu adenylation domains specifically recognized only L-Leu. Moreover, two thioesterase domains are tandemly located at the C-terminal end of ArfC. These results suggest that ArfA, ArfB, and ArfC assemble to form a unique structure. Gene disruption of arfB impaired arthrofactin production, reduced swarming activity, and enhanced biofilm formation.
ESTHER : Roongsawang_2003_Chem.Biol_10_869
PubMedSearch : Roongsawang_2003_Chem.Biol_10_869
PubMedID: 14522057
Gene_locus related to this paper: psesp-ARFC.1 , psesp-ARFC.2

Title : Role of repetitive nine-residue sequence motifs in secretion, enzymatic activity, and protein conformation of a family I.3 lipase - Kwon_2002_J.Biosci.Bioeng_93_157
Author(s) : Kwon HJ , Haruki M , Morikawa M , Omori K , Kanaya S
Ref : J Biosci Bioeng , 93 :157 , 2002
Abstract : A family I.3 lipase from Pseudomonas sp. MIS38 (PML) contains 12 repeats of a nine-residue sequence motif in the C-terminal region. To elucidate the role of these repetitive sequences, mutant proteins PML5, PML4, PML1, and PML0, in which 7, 8, 11, and all 12 of the repetitive sequences are deleted, and PMLdelta19, in which 19 C-terminal residues are truncated, were constructed. Escherichia coli DH5 cells carrying the Serratia marcescens Lip system permitted the secretion of the wild-type and all of the mutant proteins except for PMLdelta19, although they were partially accumulated in the cells in an insoluble form as well. Both the secretion level and cellular content of the proteins decreased in the order PML > PML5 > PML4 > PML1 > PML0, indicating that repetitive sequences are not required for secretion of PML but are important for its stability in the cells. All the mutant proteins were purified in a refolded form and their biochemical properties were characterized. CD spectra, the Ca2+ contents, and susceptibility to chymotryptic digestion strongly suggested that the five repetitive sequences remaining in PML5 are sufficient to form a beta-roll structure, whereas the four in PML4 are not. PML5 and PMLdelta19 showed both lipase and esterase activities, whereas PML4, PML1, and PML0 were inactive. These results suggest that the enzymatic activity of PML is not seriously affected by a deletion or truncation at the C-terminal region as long as a succession of repetitive sequences can build a beta-roll structure.
ESTHER : Kwon_2002_J.Biosci.Bioeng_93_157
PubMedSearch : Kwon_2002_J.Biosci.Bioeng_93_157
PubMedID: 16233181

Title : A phylogenomic study of the OCTase genes in Pseudomonas syringae pathovars: the horizontal transfer of the argK-tox cluster and the evolutionary history of OCTase genes on their genomes - Sawada_2002_J.Mol.Evol_54_437
Author(s) : Sawada H , Kanaya S , Tsuda M , Suzuki F , Azegami K , Saitou N
Ref : Journal of Molecular Evolution , 54 :437 , 2002
Abstract : Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999) showed that the ancestor of P. syringae had diverged into at least three monophyletic groups during its evolution. Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome. The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P. syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK-tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P. syringae pathovars (pv. actinidiae and pv. phaseolicola) from bacterial species distantly related to P. syringae and that its acquisition was quite recent (i.e., after the ancestor of P. syringae diverged into the respective pathovars). Furthermore, the results of a detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which is present within the argK-tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P. aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK-tox cluster has horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars. Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P. syringae.
ESTHER : Sawada_2002_J.Mol.Evol_54_437
PubMedSearch : Sawada_2002_J.Mol.Evol_54_437
PubMedID: 11956683
Gene_locus related to this paper: psesy-PSPTO4540

Title : Coexistence of impairment of endothelium-derived nitric oxide and platelet-derived nitric oxide in patients with coronary risk factors - Katoh_2002_Circ.J_66_837
Author(s) : Katoh A , Ikeda H , Takajo Y , Haramaki N , Murohara T , Shintani S , Kanaya S , Yokoyama S , Ueno T , Honma T , Imaizumi T
Ref : Circ J , 66 :837 , 2002
Abstract : Impairment of endothelium-derived nitric oxide (EDNO) has been demonstrated in patients with coronary risk factors in some studies, as well as impaired platelet-derived nitric oxide (PDNO) in other studies. However, no study has examined whether these impairments coexist. In 24 patients with coronary risk factors, femoral vascular endothelial function was assessed with acetylcholine (ACh: 50, 100, 200 and 400 microg/min) and endothelium-independent vascular function with nitroglycerin (NTG; 50, 100, 200 microg/min) using a Doppler flow-wire technique, as well as ADP (5 micromol/L)-induced PDNO release with an NO-specific electrode. The ACh-mediated percent change in femoral vascular resistance index (% change of FVRI) and PDNO release had a significant correlation with the number of risk factors. The ACh-mediated % change of FVRI, but not that with NTG, significantly correlated with the PDNO release. Both EDNO and PDNO bioactivities are impaired in patients with coronary risk factors and there is a common mechanism.
ESTHER : Katoh_2002_Circ.J_66_837
PubMedSearch : Katoh_2002_Circ.J_66_837
PubMedID: 12224822

Title : Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp. MIS38 - Amada_2000_Biochim.Biophys.Acta_1478_201
Author(s) : Amada K , Haruki M , Imanaka T , Morikawa M , Kanaya S
Ref : Biochimica & Biophysica Acta , 1478 :201 , 2000
Abstract : Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. MIS38 (PML) revealed that PML is a member of the lipase family I.3 and is composed of 617 amino acid residues with a calculated molecular weight of 64510. Recombinant PML (rPML) was overproduced in Escherichia coli in an insoluble form, solubilized in the presence of 8 M urea, purified in a urea-denatured form and refolded by removing urea in the presence of the Ca(2+) ion. Gel filtration chromatography suggests that this refolded protein is monomeric. rPML showed relatively broad substrate specificities and hydrolyzed glyceryl tributyrate and olive oil with comparable efficiencies. rPML was active only in the form of a holo-enzyme, in which at least 12 Ca(2+) ions bound. These Ca(2+) ions bound too tightly to be removed from the protein upon dialysis, but were removed from it upon EDTA treatment. The resultant apo-enzyme was fully active in the presence of 10 mM CaCl(2), but was inactive in the absence of the Ca(2+) ion. PML has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The mutation of Ser(207) within this motif to Ala completely inactivated PML, suggesting that Ser(207) is the active-site serine of PML.
ESTHER : Amada_2000_Biochim.Biophys.Acta_1478_201
PubMedSearch : Amada_2000_Biochim.Biophys.Acta_1478_201
PubMedID: 10825531
Gene_locus related to this paper: psesp-Q9RBY1

Title : Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1 - Mizuguchi_1999_J.Biochem_126_731
Author(s) : Mizuguchi S , Amada K , Haruki M , Imanaka T , Morikawa M , Kanaya S
Ref : J Biochem , 126 :731 , 1999
Abstract : The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.
ESTHER : Mizuguchi_1999_J.Biochem_126_731
PubMedSearch : Mizuguchi_1999_J.Biochem_126_731
PubMedID: 10502682
Gene_locus related to this paper: psesp-esthde

Title : Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis - Haruki_1999_FEBS.Lett_454_262
Author(s) : Haruki M , Oohashi Y , Mizuguchi S , Matsuo Y , Morikawa M , Kanaya S
Ref : FEBS Letters , 454 :262 , 1999
Abstract : Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.
ESTHER : Haruki_1999_FEBS.Lett_454_262
PubMedSearch : Haruki_1999_FEBS.Lett_454_262
PubMedID: 10431819
Gene_locus related to this paper: ecoli-Aes

Title : An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase - Kanaya_1998_Biochem.J_332 ( Pt 1)_75
Author(s) : Kanaya S , Koyanagi T , Kanaya E
Ref : Biochemical Journal , 332 ( Pt 1) :75 , 1998
Abstract : An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaC gene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degreesC and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coli esterase.
ESTHER : Kanaya_1998_Biochem.J_332 ( Pt 1)_75
PubMedSearch : Kanaya_1998_Biochem.J_332 ( Pt 1)_75
PubMedID: 9576853
Gene_locus related to this paper: ecoli-Aes