Ohtsuka J

References (4)

Title : Structural basis for the Ca(2+)-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190 - Miyakawa_2015_Appl.Microbiol.Biotechnol_99_4297
Author(s) : Miyakawa T , Mizushima H , Ohtsuka J , Oda M , Kawai F , Tanokura M
Ref : Applied Microbiology & Biotechnology , 99 :4297 , 2015
Abstract : A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an alpha/beta hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca(2+)-induced thermostabilization and activation of enzymes have been well explored in alpha-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca(2+) enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190(S226P)) in the Ca(2+)-bound and free states at 1.75 and 1.45 A resolution, respectively. Based on the crystallographic data, a Ca(2+) ion was coordinated by four residues within loop regions (the Ca(2+) site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca(2+) to Cut190(S226P) induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca(2+) not only stabilized a region that is flexible in the Ca(2+)-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca(2+)-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190(S226P) is activated by a conformational change in the active-site sealing residue, F106.
ESTHER : Miyakawa_2015_Appl.Microbiol.Biotechnol_99_4297
PubMedSearch : Miyakawa_2015_Appl.Microbiol.Biotechnol_99_4297
PubMedID: 25492421
Gene_locus related to this paper: sacvd-c7mve8

Title : Crystal structure of the novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 reveals a special halide-stabilizing pair and enantioselectivity mechanism - Guan_2014_Appl.Microbiol.Biotechnol_98_8573
Author(s) : Guan L , Yabuki H , Okai M , Ohtsuka J , Tanokura M
Ref : Applied Microbiology & Biotechnology , 98 :8573 , 2014
Abstract : A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. Because DatA possesses a unique Asn-Tyr pair instead of the Asn-Trp pair conserved among the subfamily members, which was proposed to keep the released halide ion stable, the structural basis for its reaction mechanism should be elucidated. Here, we determined the crystal structures of DatA and its Y109W mutant at 1.70 and 1.95 A, respectively, and confirmed the location of the active site by using its novel competitive inhibitor. The structural information from these two crystal structures and the docking simulation suggested that (i) the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for some halogenated compounds, such as 1,3-dibromopropane, mainly due to the electrostatic interaction between Trp109 and halogenated compounds and the change of substrate-binding mode caused by the interaction and (ii) the primary halide-stabilizing residue is only Asn43 in the wild-type DatA, while Tyr109 is a secondary halide-stabilizing residue. Furthermore, docking simulation using the crystal structures of DatA indicated that its enantioselectivity is determined by the large and small spaces around the halogen-binding site.
ESTHER : Guan_2014_Appl.Microbiol.Biotechnol_98_8573
PubMedSearch : Guan_2014_Appl.Microbiol.Biotechnol_98_8573
PubMedID: 24770384
Gene_locus related to this paper: agrtu-DHAA

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 : 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