MOLECULAR-BASIS FOR VANCOMYCIN RESISTANCE IN ENTEROCOCCUS-FAECIUM BM4147 - BIOSYNTHESIS OF A DEPSIPEPTIDE PEPTIDOGLYCAN PRECURSOR BY VANCOMYCIN RESISTANCE PROTEINS VANH AND VANA
MOLECULAR-BASIS FOR VANCOMYCIN RESISTANCE IN ENTEROCOCCUS-FAECIUM BM4147 - BIOSYNTHESIS OF A DEPSIPEPTIDE PEPTIDOGLYCAN PRECURSOR BY VANCOMYCIN RESISTANCE PROTEINS VANH AND VANA
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DOI：
10.1021/bi00107a007
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发表时间：
1991-10-29
期刊：
影响因子：
2.9
通讯作者：
WALSH, CT
中科院分区：
文献类型：
--
作者：
BUGG, TDH;WRIGHT, GD;WALSH, CT
屎肠球菌BM 4147的万古霉素耐药性由万古霉素耐药蛋白VanA和VanH介导。VanA是改变底物特异性的D-丙氨酸：D-丙氨酸连接酶[Bugg，T. D. H、Dutka-Malen，S.，亚瑟先生，Courvalin，P.，&沃尔什，C. T.（1991）Biochemistry 30，2017-2021]，而VanH的序列与α-酮酸脱氢酶的序列相关[亚瑟，M.，Molinas角，Dutka-Malen，S.，& Courvalin，P.（1991）Gene（submitted）].我们报告纯化VanH的同质性，表征为D-特异性α-酮酸脱氢酶，并与肠膜明串珠菌和Lactobacillus leichmanii的D-乳酸脱氢酶比较。发现VanA催化D-丙氨酸和VanH的D-羟基酸产物之间的酯键形成，最佳底物为D-2-羟基丁酸酯（K（m）= 0.60 mM）。然后通过D-Ala-D-Ala添加来自大肠杆菌的酶或通过来自大肠杆菌的粗提物将VanA产物D-丙氨酰-D-2-羟基丁酸酯掺入UDPMurNAc-五肽聚糖前体。faecium BM 4147.合成的修饰肽聚糖类似物N-乙酰基-D-丙氨酰-D-2-羟基丁酸酯的万古霉素结合常数（K（d）> 73 mM）比N-乙酰基-D-丙氨酰-D-丙氨酸的结合常数（K（d）= 54 μ M）高> 1000倍，部分原因是万古霉素-靶复合物中氢键的破坏，从而为高水平万古霉素抗性提供了分子理论基础。
Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (K(m) = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (K(d) > 73 mM) was > 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (K(d) = 54-mu-M), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.