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- Title
Isolation and molecular characterization of a noveld-hydantoinase from Jannaschia sp. CCS1.
- Authors
Yuanheng Cai; Trodler, Peter; Jiang, Shimin; Weiwen Zhang; Yan Wu; Yinhua Lu; Sheng Yang; Weihong Jiang
- 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 HYDJs, was identified from Jannaschia sp. CCS1 and overexpressed in Escherichia coli. The specific activity of HYDJs ond,l- p-hydroxyphenylhydantoin as the substrate was three times higher than that of the hydantoinase originating from Burkholderia pickettii (HYDBp) 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 HYDJs were 7.6 and 50 °C respectively, similar to those of HYDBp. Kinetic analysis showed that HYDJs has a higher kcat value ond,l- p-hydroxyphenylhydantoin than HYDBp does. Homology modeling and substrate docking analyses of HYDJs and HYDBp were performed, and the results revealed an enlarged substrate binding pocket in HYDJs, which may allow better access of substrates to the catalytic centre and could account for the increased specific activity of HYDJs. Three amino acid residues critical for HYDJs activity, Phe63, Leu92 and Phe150 were also identified by substrate docking and site-directed mutagenesis. Application of this high-specific activity HYDJs could improve the industrial production of optically pure amino acids, such asd- 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.
- Subjects
AMINO acids; ANTIBIOTICS; ESCHERICHIA coli; MUTAGENESIS; BIOCHEMISTRY
- Publication
FEBS Journal, 2009, Vol 276, Issue 13, p3575
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2009.07077.x