title: Isolation and molecular characterization of a novel D-hydantoinase from Jannaschia sp. CCS1.
abstract: Hydantoinases (HYDs) are important enzymes for industrial production of optically pure amino acids, which are widely used as precursors for various semi-synthetic antibiotics. By a process coupling genomic data mining with activity screening, a new hydantoinase, tentatively designated HYD(Js), was identified from Jannaschia sp. CCS1 and overexpressed in Escherichia coli. The specific activity of HYD(Js) on D,L-p-hydroxyphenylhydantoin as the substrate was three times higher than that of the hydantoinase originating from Burkholderia pickettii (HYD(Bp)) that is currently used in industry. The enzyme obtained was a homotetramer with a molecular mass of 253 kDa. The pH and temperature optima for HYD(Js) were 7.6 and 50 degrees C respectively, similar to those of HYD(Bp). Kinetic analysis showed that HYD(Js) has a higher k(cat) value on D,L-p-hydroxyphenylhydantoin than HYD(Bp) does. Homology modeling and substrate docking analyses of HYD(Js) and HYD(Bp) were performed, and the results revealed an enlarged substrate binding pocket in HYD(Js), which may allow better access of substrates to the catalytic centre and could account for the increased specific activity of HYD(Js). Three amino acid residues critical for HYD(Js) activity, Phe63, Leu92 and Phe150 were also identified by substrate docking and site-directed mutagenesis. Application of this high-specific activity HYD(Js) could improve the industrial production of optically pure amino acids, such as D-p-hydroxyphenylglycine. Moreover, the structural analysis also provides new insights on enzyme-substrate interaction, which shed light on engineering of hydantoinases for high catalytic activity.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yuanheng
authors.initials: Y
authors.lastname: Cai
authors.forename: Peter
authors.initials: P
authors.lastname: Trodler
authors.forename: Shimin
authors.initials: S
authors.lastname: Jiang
authors.forename: Weiwen
authors.initials: W
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.forename: Yinhua
authors.initials: Y
authors.lastname: Lu
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Weihong
authors.initials: W
authors.lastname: Jiang
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020134:Catalytic Domain; D018835:Chaperonin 10 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D041902:Rhodobacteraceae / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D000581:Amidohydrolases; C020047:dihydropyrimidinase