Lessard I A, Pratt S D, McCafferty D G, Bussiere D E, Hutchins C, Wanner B L, Katz L, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Chem Biol. 1998 Sep;5(9):489-504. doi: 10.1016/s1074-5521(98)90005-9.
Vancomycin-resistant enterococci are pathogenic bacteria that have altered cell-wall peptidoglycan termini (D-alanyl-D-lactate [D-Ala-D-lactate] instead of D-alanyl-D-alanine [D-Ala-D-Ala]), which results in a 1000-fold decreased affinity for binding vancomycin. The metallodipeptidase VanX (EntVanX) is key enzyme in antibiotic resistance as it reduces the cellular pool of the D-Ala-D-Ala dipeptide.
A bacterial genome search revealed vanX homologs in Streptomyces toyocaensis (StoVanX), Escherichia coli (EcoVanX), and Synechocystis sp. strain PCC6803 (SynVanX). Here, the D,D-dipeptidase catalytic activity of all three VanX homologs is validated, and the catalytic efficiencies and diastereoselectivity ratios for dipeptide cleavage are reported. The ecovanX gene is shown to have an RpoS (sigma(s))-dependent promoter typical of genes turned on in stationary phase. Expression of ecovanX and an associated cluster of dipeptide permease genes permitted growth of E. coli using D-Ala-D-Ala as the sole carbon source.
The key residues of the EntVanX active site are strongly conserved in the VanX homologs, suggesting their active-site topologies are similar. StoVanX is a highly efficient D-Ala-D-Ala dipeptidase; its gene is located in a vanHAX operon, consistent with a vancomycin-immunity function. StoVanX is a potential source for the VanX found in gram-positive enterococci. The catalytic efficiencies of D-Ala-D-Ala hydrolysis for EcoVanX and SynVanX are 25-fold lower than for EntVanX, suggesting they have a role in cell-wall turnover. Clustered with the ecovanX gene is a putative dipeptide permease system that imports D-Ala-D-Ala into the cell. The combined action of EcoVanX and the permease could permit the use of D-Ala-D-Ala as a bacterial energy source under starvation conditions.
耐万古霉素肠球菌是一种病原菌，其细胞壁肽聚糖末端发生了改变（由D - 丙氨酰 - D - 丙氨酸[D - Ala - D - Ala]变为D - 丙氨酰 - D - 乳酸[D - Ala - D - 乳酸]），这导致其与万古霉素结合的亲和力降低了1000倍。金属二肽酶VanX（EntVanX）是抗生素耐药性中的关键酶，因为它能减少细胞内D - Ala - D - Ala二肽的总量。
细菌基因组搜索在丰田链霉菌（StoVanX）、大肠杆菌（EcoVanX）和聚球藻属PCC6803菌株（SynVanX）中发现了vanX同源物。在此，验证了所有三种VanX同源物的D，D - 二肽酶催化活性，并报告了二肽裂解的催化效率和非对映选择性比率。ecovanX基因被证明具有典型的在稳定期开启的基因所具有的依赖RpoS（σ(s)）的启动子。ecovanX和相关的二肽通透酶基因簇的表达使大肠杆菌能够利用D - Ala - D - Ala作为唯一碳源生长。
EntVanX活性位点的关键残基在VanX同源物中高度保守，表明它们的活性位点拓扑结构相似。StoVanX是一种高效的D - Ala - D - Ala二肽酶；其基因位于vanHAX操纵子中，与万古霉素免疫功能一致。StoVanX是革兰氏阳性肠球菌中发现的VanX的潜在来源。EcoVanX和SynVanX对D - Ala - D - Ala水解的催化效率比EntVanX低25倍，表明它们在细胞壁周转中起作用。与ecovanX基因聚集在一起的是一个假定的二肽通透酶系统，该系统将D - Ala - D - Ala导入细胞。EcoVanX和通透酶的联合作用可能使大肠杆菌在饥饿条件下能够利用D - Ala - D - Ala作为细菌能量来源。
