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.
ACS Chem Biol. 2019 May 17;14(5):975-978. doi: 10.1021/acschembio.9b00159. Epub 2019 Apr 17.
We recently revealed that a previously unknown pathway for peptidoglycan biosynthesis operates in some microorganisms, including Xanthomonas oryzae. It involves two enzymes, MurD2 and MurL, which catalyze the ligation of l-glutamate (l-Glu) to UDP- N-acetylmuramic acid-l-alanine and the epimerization of the terminal l-Glu of the product, respectively. MurD2 of X. oryzae possesses a 26% identity with MurD of Escherichia coli (MurD), which ligates d-Glu to UDP- N-acetylmuramic acid-l-alanine. To understand how X. oryzae MurD2 recognizes the isomer substrate, we estimated its structure based on that of MurD during docking simulations. Several amino acid residues, which may be responsible for l-Glu recognition, were replaced with their corresponding amino acid residues in MurD. Consequently, we obtained a mutated MurD2 enzyme that contained two amino acid substitutions and accepted only d-Glu as the substrate. We next tried to convert the substrate specificity of MurD using the same strategy, but the mutant enzyme still accepted only d-Glu. Then, MurD of Streptococcus mutans (MurD), which possesses the key amino acid residue for l-Glu recognition identified in MurD2, was used for random screenings of mutant enzymes accepting l-Glu. We obtained a mutated MurD that had one amino acid substitution and slightly accepted l-Glu. A mutated MurD possessing the corresponding one amino acid substitution also accepted l-Glu. Thus, we revealed that a few amino acid residues in MurD/MurD2 might control the acceptability of substrates with different stereochemistries.
我们最近揭示了一个以前未知的肽聚糖生物合成途径在某些微生物中起作用，包括稻黄单胞菌。它涉及两种酶，MurD2 和 MurL，它们分别催化 l-谷氨酸（l-Glu）与 UDP-N-乙酰胞壁酸-l-丙氨酸的连接和产物末端 l-Glu的差向异构化。稻黄单胞菌的 MurD2 与大肠杆菌的 MurD （MurD）具有 26%的同一性，后者将 d-Glu 连接到 UDP-N-乙酰胞壁酸-l-丙氨酸上。为了了解稻黄单胞菌 MurD2 如何识别异构底物，我们根据 MurD 的结构在对接模拟中估计了其结构。可能负责识别 l-Glu 的几个氨基酸残基被替换为 MurD 中的相应氨基酸残基。结果，我们获得了一种突变 MurD2 酶，其中包含两个氨基酸取代，仅接受 d-Glu 作为底物。接下来，我们尝试使用相同的策略改变 MurD 的底物特异性，但突变酶仍然只接受 d-Glu。然后，使用具有在 MurD2 中鉴定出的 l-Glu 识别关键氨基酸残基的 Streptococcus mutans MurD （MurD）进行接受 l-Glu 的突变酶的随机筛选。我们获得了一个具有一个氨基酸取代的突变 MurD，它略微接受 l-Glu。具有相应一个氨基酸取代的突变 MurD 也接受 l-Glu。因此，我们揭示 MurD/MurD2 中的几个氨基酸残基可能控制具有不同立体化学的底物的可接受性。
