细菌对万古霉素的耐药性：从格氏肠球菌中过量表达、纯化及鉴定作为D-丙氨酰-D-丝氨酸连接酶的VanC2
Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase.
作者信息
Park I S, Lin C H, Walsh C T
机构信息
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
出版信息
Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10040-4. doi: 10.1073/pnas.94.19.10040.
Abstract
The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in D-Ala-D-Ser instead of D-Ala-D-Ala, it has been predicted that the VanC ligase would be a D-Ala-D-Ser rather than a D-Ala-D-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent D-Ala-D-Ser ligase activity. The kcat/Km2 (Km2 = Km for D-Ser or C-terminal D-Ala) ratio for D-Ala-D-Ser/D-Ala-D-Ala dipeptide formation is 270/0.69 for a 400-fold selection against D-Ala in the C-terminal position. VanC2 also has substantial D-Ala-D-Asn ligase activity (kcat/Km2 = 74 mM-1min-1).
摘要
鹑鸡肠球菌和格氏肠球菌表现出肠球菌对万古霉素临床耐药的VanC表型。基于对以D-Ala-D-Ser而非D-Ala-D-Ala结尾的细胞前体UDP-N-乙酰胞壁酸五肽中间体的检测，据预测VanC连接酶将是一种D-Ala-D-Ser连接酶而非D-Ala-D-Ala连接酶。在大肠杆菌中过量表达格氏肠球菌ATCC 25788的vanC2基因并将其纯化至同质，从而证明了ATP依赖性D-Ala-D-Ser连接酶活性。对于C末端位置对D-Ala有400倍选择性的D-Ala-D-Ser/D-Ala-D-Ala二肽形成，kcat/Km2（Km2 = D-Ser或C末端D-Ala的Km）比值为270/0.69。VanC2也具有显著的D-Ala-D-Asn连接酶活性（kcat/Km2 = 74 mM-1min-1）。
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