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 HYDJs, was identified from Jannaschia sp. CCS1 and overexpressed in Escherichia coli. The specific activity of HYDJs on d,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 HYD Js has a higher kcat value on d,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 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. © 2009 FEBS.