Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725 |
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| Authors: | Yi-Chia Kuan Chao-Hung Kao Chao-Hsien Chen Chang-Chih Chen Hui-Yu Hu Wen-Hwei Hsu |
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| Institution: | aInstitute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan;bDepartment of Biotechnology, Hung Kuang University, Taichung 433, Taiwan;cDepartment of Medical Laboratory Science and Biotechnology, China Medical University, Taichung 404, Taiwan;dSimpson Biotech Co., Ltd., Taoyuan County 333, Taiwan;eDepartment of Food Science and Technology, Hung Kuang University, Taichung 433, Taiwan |
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| Abstract: | A lysine racemase gene (lyr) that consisted of an open reading frame of 1224-bp and encoded a protein with a calculated molecular mass of 45 kDa was cloned from the Proteus mirabilis BCRC10725 and expressed in Escherichia coli BL21(DE3). The purified His6-tagged Lyr was most active towards lysine, exhibiting a specific activity of 2828 ± 97 U/mg. This enzyme also racemized arginine with a specific activity of 568 ± 28 U/mg but not other amino acids. The optimal conditions for Lyr activity to l-lysine were pH 8.0–9.0 and 50 °C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxylamine and was partially restored by the addition of pyridoxal 5′-phosphate. The S394 residue of Lyr was subjected to site-directed mutagenesis. The arginine racemization activities of the S394Y, S394N, S394C and S394T variant proteins were increased by 1.5–1.8 fold compared to the wild-type Lyr, indicating that the S394 residue played a crucial role in determining the preference of Lyr to lysine and arginine. |
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| Keywords: | Proteus mirabilis Lysine racemase Lysine racemization Arginine racemization PLP-dependent enzyme Substrate specificity |
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