Wu Z, Wright G D, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1995 Feb 28;34(8):2455-63. doi: 10.1021/bi00008a008.
Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)
vanR、vanS、vanH、vanA和vanX。四种基因产物的功能和机制已经明确，VanR/S负责信号转导和转录调控，VanH/A负责合成D-丙氨酰-D-乳酸。但直到最近，第五种基因产物VanX的功能一直未知，当时雷诺兹及其同事在VanX过量生产者的粗提物中发现了D，D-二肽酶活性[雷诺兹，P.E.等人（1994年）。分子微生物学。13，1065 - 1070]。我们在此报告VanX在大肠杆菌中的表达及其纯化至同质状态。VanX已被鉴定为一种金属激活的D，D-二肽酶，最佳pH范围为7 - 9。在没有二价金属的情况下，D-丙氨酰-D-丙氨酸的kcat和Km分别测定为4.7 s-1和1 mM。然而，在金属阳离子存在的情况下，kcat可高达788 s-1。VanX无法水解D-丙氨酰-D-乳酸，即肽聚糖中导致万古霉素耐药性的取代部分，这不仅是因为结合亲和力低（Ki估计为242 mM），还因为kcat小于0.005 s-1。VanX对D-丙氨酰-D-丙氨酸与D-丙氨酰-D-乳酸水解的催化效率相差超过10^5倍，使得D-丙氨酰-D-乳酸保持完整，以便随后掺入肽聚糖中。（摘要截短至250字）
