Tài liệu Báo cáo khoa học: Isolation and molecular characterization of a novel D-hydantoinase from

Isolation and molecular characterization of a novel

D-hydantoinase from Jannaschia sp. CCS1

Yuanheng Cai1

, Peter Trodler2

, Shimin Jiang1

, Weiwen Zhang3

, Yan Wu1

, Yinhua Lu1

, Sheng Yang1

and Weihong Jiang1,4

1 Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese

Academy of Sciences, Shanghai, China

2 Institute of Technical Biochemistry, University of Stuttgart, Germany

3 Center for Ecogenomics, Biodesign Institute, Arizona State University, Tempe, AZ, USA

4 Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China

Optically pure d- or l-amino acids are used as inter￾mediates in several industries. d-amino acids are

involved in the synthesis of antibiotics, pesticides,

sweeteners and other biologically active peptides.

l-amino acids are used as feed and food additives, as

intermediates for pharmaceuticals, cosmetics and pesti￾cides, and as chiral compounds in organic synthesis [1–4].

Among them, d-p-hydroxyphenylglycine (d-p-HPG)

attracts the most attention as it can be used as the side

chain for production of semi-synthetic b-lactam antibi￾otics, such as amoxicillins and cephalosporins [2].

There are currently two main approaches used to

Keywords

hydantoinase; Jannaschia sp. CCS1;

saturated mutagenesis; structural analysis;

substrate binding pocket

Correspondence

W. Jiang, Key Laboratory of Synthetic

Biology, Institute of Plant Physiology and

Ecology, Shanghai Institutes for Biological

Sciences, Chinese Academy of Sciences,

Shanghai, 200032, China

Fax: +86 21 54924015

Tel: +86 21 54924172

E-mail: [email protected]

(Received 4 February 2009, revised 15 April

2009, accepted 27 April 2009)

doi:10.1111/j.1742-4658.2009.07077.x

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 respec￾tively, similar to those of HYDBp. Kinetic analysis showed that HYDJs 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 cata￾lytic 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 muta￾genesis. Application of this high-specific activity HYDJs could improve the

industrial production of optically pure amino acids, such as d-p-hydroxy￾phenylglycine. Moreover, the structural analysis also provides new insights

on enzyme–substrate interaction, which shed light on engineering of hydan￾toinases for high catalytic activity.

Abbreviations

DCase, N-carbamoyl-D-amino acid amidohydrolase; DHU, dihydrouracil; D-p-HPG, D-p-hydroxyphenylglycine; D,L-p-HPH, D,L-p￾hydroxyphenylhydantoin; HDT, hydantoin; HYD, hydantoinase; HYDBp, hydantoinase from Burkholderia pickettii; HYDJs, hydantoinase from

Jannaschia sp. CCS1; PDB, protein data bank; SGLs, stereochemistry gate loops.

FEBS Journal 276 (2009) 3575–3588 ª 2009 The Authors Journal compilation ª 2009 FEBS 3575