Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids
Identification of a novel amino acid racemase from a hyperthermophilic archaeon Pyrococcus horikoshii OT-3 induced by D-amino acids
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DOI：
10.1007/s00726-015-2001-6
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发表时间：
2015-08-01
期刊：
影响因子：
3.5
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Kawakami, Ryushi;Ohmori, Taketo;Ohshima, Toshihisa
迄今为止，很少有报道分析极端嗜热古菌生长所需的氨基酸。我们在此发现，超嗜热古菌Pyrococcus horikoshii OT-3在培养基中需要Thr、Leu、瓦尔、Phe、Tyr、Trp、His和Arg来生长，并且在缺乏Met或Ile的培养基中显示缓慢生长。这在很大程度上对应于其基因组中与氨基酸生物合成相关的基因的存在或缺失，尽管也有例外。通过添加Met、Leu、瓦尔、allo-Ile、Phe、Tyr、Trp和Arg的d-异构体，氨基酸需求量显著损失。使用C-14标记的d-Trp的示踪剂分析表明，培养基中的d-Trp被用作细胞中的蛋白质组分，表明存在d-氨基酸代谢酶。在堀越氏原球茎的粗提物中检测到5 '-磷酸吡哆醛（PLP）依赖的消旋酶对Met、Leu和Phe的活性，并且在添加d-氨基酸，尤其是d-allo-Ile的培养基中生长的细胞中增强。从堀越氏毕赤酵母细胞中纯化的消旋酶编码基因被缩小到一个开放的阅读框架，重组酶对几种氨基酸表现出PLP依赖的消旋酶活性，包括Met，Leu和Phe，但不包括Pro，Asp和Glu。这是第一份报告显示存在于超嗜热古菌的PLP依赖性氨基酸消旋酶具有广泛的底物特异性，这可能是负责利用d-氨基酸的增长。
To date, there have been few reports analyzing the amino acid requirement for growth of hyperthermophilic archaea. We here found that the hyperthermophilic archaeon Pyrococcus horikoshii OT-3 requires Thr, Leu, Val, Phe, Tyr, Trp, His and Arg in the medium for growth, and shows slow growth in medium lacking Met or Ile. This largely corresponds to the presence, or absence, of genes related to amino acid biosynthesis in its genome, though there are exceptions. The amino acid requirements were dramatically lost by addition of d-isomers of Met, Leu, Val, allo-Ile, Phe, Tyr, Trp and Arg. Tracer analysis using C-14-labeled d-Trp showed that d-Trp in the medium was used as a protein component in the cells, suggesting the presence of d-amino acid metabolic enzymes. Pyridoxal 5'-phosphate (PLP)-dependent racemase activity toward Met, Leu and Phe was detected in crude extract of P. horikoshii and was enhanced in cells grown in the medium supplemented with d-amino acids, especially d-allo-Ile. The gene encoding the racemase was narrowed down to one open reading frame on the basis of enzyme purification from P. horikoshii cells, and the recombinant enzyme exhibited PLP-dependent racemase activity toward several amino acids, including Met, Leu and Phe, but not Pro, Asp or Glu. This is the first report showing the presence in a hyperthermophilic archaeon of a PLP-dependent amino acid racemase with broad substrate specificity that is likely responsible for utilization of d-amino acids for growth.