Reynolds P E, Depardieu F, Dutka-Malen S, Arthur M, Courvalin P
Department of Biochemistry, University of Cambridge, UK.
Mol Microbiol. 1994 Sep;13(6):1065-70. doi: 10.1111/j.1365-2958.1994.tb00497.x.
Cloning and nucleotide sequencing indicated that transposon Tn1546 from Enterococcus faecium BM4147 encodes a 23,365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2-2 derivative producing VanH, VanA and VanX accumulated mainly UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac (pentadepsipeptide) and small amounts of UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D-dipeptidase that hydrolysed D-Ala-D-Ala. Pentadepsipeptide, pentapeptide and D-Ala-D-Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D-dipeptidase that hydrolyses D-Ala-D-Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D-Ala-D-Ala-containing peptidoglycan precursors at the cell surface.
克隆和核苷酸测序表明，来自粪肠球菌BM4147的转座子Tn1546编码一种23365 Da的蛋白质VanX，它是糖肽抗性所必需的。vanX基因位于编码合成二肽D-丙氨酰-D-乳酸（D-Ala-D-Lac）的VanA连接酶和VanH脱氢酶的基因下游。在瑞莫拉宁存在的情况下，一株产生VanH、VanA和VanX的粪肠球菌JH2-2衍生物主要积累UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Lac（五肽二肽）和少量UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala（五肽），比例为49:1。vanX的插入失活导致五肽合成增加，从而使五肽二肽与五肽的比例变化至小于1:1。vanX在粪肠球菌和大肠杆菌中的表达导致产生一种能水解D-Ala-D-Ala的D,D-二肽酶。五肽二肽、五肽和D-Ala-D-Lac不是该酶的底物。这些结果表明，VanX是产生水解D-Ala-D-Ala的D,D-二肽酶所必需的，从而防止五肽合成以及随后糖肽与细胞表面含D-Ala-D-Ala的肽聚糖前体结合。
