Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis
Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis
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DOI：
10.1093/bbb/zbab078
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发表时间：
2021-05-04
影响因子：
1.6
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Kawakami, Ryushi;Kinoshita, Chinatsu;Ohshima, Toshihisa
来自超嗜热古菌Thermococcus litoralis DSM 5473的OCC_10945基因产物的氨基酸序列（最初注释为γ-氨基丁酸转氨酶）与来自Pyrococcus horikoshii的未表征的吡哆醛5 '-磷酸（PLP）依赖性氨基酸消旋酶的氨基酸序列高度相似。OCC_10945酶在大肠杆菌中通过与伴侣蛋白共表达成功过表达。纯化的酶表现出PLP依赖性的氨基酸消旋酶活性，主要是对蛋氨酸和亮氨酸。虽然PLP有助于酶的稳定性，但它仅与该酶松散结合。几种金属离子，包括Co ~（2+）和Zn ~（2+），以及非底物氨基酸如L-Arg和L-Lys强烈抑制酶活性。这些结果表明，潜在的PLP结合和底物识别机制在这种酶是显着不同的其他古细菌和细菌的氨基酸消旋酶。这是第一次描述一种新的PLP依赖的氨基酸消旋酶与适度的底物特异性在超嗜热古菌。[图形]。
The amino acid sequence of the OCC_10945 gene product from the hyperthermophilic archaeon Thermococcus litoralis DSM5473, originally annotated as gamma-aminobutyrate aminotransferase, is highly similar to that of the uncharacterized pyridoxal 5'-phosphate (PLP)-dependent amino acid racemase from Pyrococcus horikoshii. The OCC_10945 enzyme was successfully overexpressed in Escherichia coli by coexpression with a chaperone protein. The purified enzyme demonstrated PLP-dependent amino acid racemase activity primarily toward Met and Leu. Although PLP contributed to enzyme stability, it only loosely bound to this enzyme. Enzyme activity was strongly inhibited by several metal ions, including Co2+ and Zn2+, and nonsubstrate amino acids such as L-Arg and L-Lys. These results suggest that the underlying PLP-binding and substrate recognition mechanisms in this enzyme are significantly different from those of the other archaeal and bacterial amino acid racemases. This is the first description of a novel PLP-dependent amino acid racemase with moderate substrate specificity in hyperthermophilic archaea.[GRAPHICS].