Graduate School of Chemical Sciences and Engineering, Hokkaido University , N13 & W8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan.
Graduate School of Engineering, Hokkaido University , N13 & W8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan.
J Am Chem Soc. 2017 Mar 29;139(12):4243-4245. doi: 10.1021/jacs.7b01221. Epub 2017 Mar 16.
d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.
d-谷氨酸（Glu）由 Glu 外消旋酶或 d-氨基酸转氨酶提供，用于微生物的肽聚糖生物合成。比较基因组学表明，包括稻黄单胞菌在内的一些微生物可能没有这些基因的直系同源物。我们以 d-谷氨酸营养缺陷型大肠杆菌突变体为宿主，以稻黄单胞菌为 DNA 供体，进行了鸟枪法克隆实验。我们获得了互补基因 XOO_1319 和 XOO_1320，它们分别被注释为一个假定蛋白和 MurD（UDP-MurNAc-l-Ala-d-Glu 合成酶）。通过详细的体外分析，我们揭示了 XOO_1320 是一种将 l-Glu 连接到 UDP-MurNAc-l-Ala 的酶，提供了 MurD 利用 l-Glu 的第一个例子，而 XOO_1319 是一种在 ATP 和 Mg 存在下催化产物末端 l-Glu 差向异构化的新型酶。我们研究了 XOO_1319 直系同源物的发生情况，发现它存在于一些微生物类别中，包括致病菌。
