Distribution of Amino Acid Racemases and D-Amino Acids in Archaea and Their Function

我们已经发现，D-氨基酸普遍存在于超嗜热古菌，热球菌和热球菌菌株。特别是天冬氨酸几乎完全外消旋化。从嗜热球菌KS-8菌株中克隆了天冬氨酸消旋酶的全长基因，并在大肠杆菌中表达。重组蛋白在70 ℃下具有较高的天冬氨酸消旋酶活性和微量的谷氨酸消旋酶活性。在超嗜热古菌的全基因组序列中发现了几个天冬氨酸消旋酶同源基因。其中，Pyrococcus horikoshii的两个天冬氨酸消旋酶同源物（PH 0670，PH 1733）和Archaeoglobus fulgidus的一个同源物（AF 1422）在大肠杆菌中表达。所有这些都是高表达的，并且容易通过热处理和层析纯化。其中，只有PH 0670表现出消旋酶活性。Km和Kcat值分别估计为3.6 E-3（M）和4.2 E-10（mol/sec）。而PH 1733和AF 1422则没有天冬氨酸消旋酶活性。它们与大肠杆菌的OrfX高度同源的事实表明它们具有二氨基庚二酸差向异构酶活性。然而，他们不能差向异构化二氨基庚二酸的对映异构体。我们可以制备PH 0670、PH 1733、AF 1422和古生菌二氨基庚二酸差向异构酶的晶体。此外，我们还发现在嗜酸热原体中天冬氨酸也是高度外消旋的。

We have revealed that D-amino acids exist ubiquitously in hyperthermophilic archaea, Pyrococcus and Thermococcus strains. Especially, aspartic acid was almost completely racemized. The full length gene of aspartate racemase was cloned from Thermococcus strain KS-8, and it was expressed in E.coli. The recombinant protein exhibited high aspartate racemase activity at 70 ℃ and trace glutamate racemase activity. Several aspartate racemase homologue genes were found in the total genomic sequences of hyperthermophilic archaea. Among them, two aspartate racemase homologues of Pyrococcus horikoshii (PH0670, PH 1733), and one homologue of Archaeoglobus fulgidus (AF l422) were expressed in E.coli. All of them were highly expressed, and easily purified by heat treatment and chromatography. Among them, only PH0670 exhibited asparate racemase activity. The Km and Kcat values were estimated to be 3.6 E-3 (M) and 4.2E-10 (mol/sec), respectively. On the contrary, PH 1733 and AF1422 showed no aspartate racemase activity. The fact that they are highly homologous to 0rfX of E.coli suggested that they have diaminopimelate epimerase activity. However, they could not epimerize an enantiomer of diaminopimelate. We could have make crystals of PH0670, PH1733, AF1422 and Archaeoglobus diaminopimelate epimerase. In addition, we have found that aspartic acid in Themoplasma acidophilum was also highly racemized.