Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species
Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species
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DOI：
10.1128/jb.00709-13
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发表时间：
2013-11-01
影响因子：
3.2
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Mutaguchi, Yuta;Ohmori, Taketo;Ohshima, Toshihisa
在乳酸菌Lactobacillus otakiensis JCM 15040的生长培养基中观察到D-亮氨酸、D-别异亮氨酸和D-缬氨酸的积累，并从细胞中纯化并鉴定了负责的消旋酶。纯化的酶的N-末端氨基酸序列为GKLDKASKLI，这与来自布氏乳杆菌的推定的γ-氨基丁酸氨基转移酶的序列一致。从L. buchneri JCM 1115在重组大肠杆菌中表达，然后纯化至均一。该酶催化广谱非极性氨基酸的外消旋化。特别是，它以高速率催化L-异亮氨酸向D-别异亮氨酸和D-别异亮氨酸向L-异亮氨酸的差向异构化。相反，该酶没有γ-氨基丁酸转氨酶活性。亚基和天然酶的相对分子质量估计分别为约49 kDa和200 kDa，表明该酶是由四个等分子量的亚基组成。该酶对L-异亮氨酸的Km值和Vmax值分别为5.00 mM和153 μ mol·min（-1）·mg（-1），对D-别异亮氨酸的Km值和Vmax值分别为13.2 mM和286 μ mol·min（-1）·mg（-1）。羟胺和其他吡哆醛5 '-磷酸依赖性酶的抑制剂完全阻断了酶的活性，表明该酶需要吡哆醛5'-磷酸作为辅酶。这是氨基酸消旋酶特异性催化非极性氨基酸在C-2位的外消旋化的第一个证据。
Accumulation of D-leucine, D-allo-isoleucine, and D-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative gamma-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative gamma-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of L-isoleucine to D-allo-isoleucine and D-allo-isoleucine to L-isoleucine. In contrast, the enzyme showed no gamma-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K-m and V-max values of the enzyme for L-isoleucine were 5.00 mM and 153 mu mol.min(-1).mg(-1), respectively, and those for D-allo-isoleucine were 13.2 mM and 286 mu mol.min(-1).mg(-1), respectively. Hydroxylamine and other inhibitors of pyridoxal 5'-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5'-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.