Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongen
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ORIGINAL PAPER
Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 Zhangwei Xue • Yi Hu • Shujing Xu • Kouhei Ohnishi • Yanhe Ma • Jiansong Ju Baohua Zhao
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Received: 17 February 2012 / Accepted: 12 May 2013 / Published online: 24 May 2013 Ó Springer Japan 2013
Abstract A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The fulllength gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His6-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters Km and Vmax of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 lmol min-1 for -1 L-alanine, and 9.95 mM and 702.6 lmol min for D-alanine, respectively. Enzymatic assays showed that the Communicated by F. Robb. Z. Xue and Y. Hu contributed equally to this study. Z. Xue Y. Hu S. Xu J. Ju (&) B. Zhao (&) College of Life Science, Hebei Normal University, Shijiazhuang 050024, China e-mail: [email protected] B. Zhao e-mail: [email protected] K. Ohnishi Institute of Molecular Genetics, Kochi University, Kochi 783-8502, Japan Y. Ma State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China e-mail: [email protected]
activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2. Keywords Alanine racemase Entryway corridor Mutant Pyridoxal 50 -phosphate Thermoanaerobacter tengcongensis
Introduction Alanine racemase (EC 5.1.1.1) belongs to the type III group of pyridoxal 50 -phosphate (PLP)-dependent enzyme with the function for catalyzing the interconversion from L-alanine to D-alanine. In most bacteria, D-alanine is incorporated into peptidoglycan and converted from L-alanine by this specific enzyme. So far, two independent alanine racemase genes such as alr and dadX encoded for the biosynthesis and catabolism of alanine racemase have been identified and characterized, respectively. The alr gene is expressed in cells for peptidoglycan biosynthesis, while dadX gene expression is induced when cells are grown in medium with L- or D-alanine at high concentrations (Lobocka et al. 1994; Zhang et al. 2007), so that the cells are able to use L-alanine as the carbon and energy source (McFall and Newman 1996). Some bacteria have a single alanine racemase gene, whereas others have two (alr and dadX). For example, there are two
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