Characterization and modelling of VanT:: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174
Characterization and modelling of VanT:: a novel, membrane-bound, serine racemase from vancomycin-resistant Enterococcus gallinarum BM4174
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DOI：
10.1046/j.1365-2958.1999.01294.x
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发表时间：
1999-03-01
影响因子：
3.6
通讯作者：
Reynolds, PE
中科院分区：
文献类型：
--
作者：
Arias, CA;Martín-Martinez, M;Reynolds, PE
对鸡肠球菌 BM4174 中 vanXY(c) 基因下游区域的序列测定揭示了一个开放阅读框，命名为 vanT，它编码 698 个氨基酸的多肽，其氨基末端结构域包含 10 个预测的跨膜片段。该蛋白的 C 端结构域含有高度保守的磷酸吡哆醛附着位点，这是丙氨酸消旋酶的典型特征。该蛋白在大肠杆菌中过表达，超声处理的细胞离心后，在细胞膜中检测到丝氨酸消旋酶活性，但在细胞质部分中未检测到丝氨酸消旋酶活性，而丙氨酸消旋酶活性几乎完全位于细胞质中。当蛋白质过表达为缺乏预测的跨膜结构域的多肽时，在细胞质中检测到丝氨酸消旋酶活性。丝氨酸消旋酶活性被D-环丝氨酸部分抑制(64%)，而宿主丙氨酸消旋酶活性几乎完全被抑制(97%)。在组成型耐万古霉素鸡肠杆菌 BM4174 的膜制剂中也检测到丝氨酸消旋酶活性，但在 BM4175 中未检测到，其中 vanC-1 D-Ala:D-Ser 连接酶基因的插入失活可能对 vanXY(c) 和 vanT 基因的表达产生极性影响。推导的 C 末端结构域的比较模型基于 VanT 与来自嗜热脂肪芽孢杆菌的 Alr 丙氨酸消旋酶的比对。模型显示，Alr活性位点的几乎所有关键氨基酸在VanT中都是保守的，这表明VanT的C端结构域很可能采用与Alr相似的三维结构，并且该蛋白可能以二聚体形式存在。这些结果表明，表达 VanC 表型的万古霉素抗性肠球菌中肽聚糖合成的 D-丝氨酸来源涉及膜结合丝氨酸消旋酶将 L-丝氨酸外消旋为 D-丝氨酸。
Sequence determination of a region downstream from the vanXY(c) gene in Enterococcus gallinarum BM4174 revealed an open reading frame, designated vanT, that encodes a 698-amino-acid polypeptide with an amino-terminal domain containing 10 predicted transmembrane segments. The protein contained a highly conserved pyridoxal phosphate attachment site in the C-terminal domain, typical of alanine racemases. The protein was overexpressed in Escherichia coli, and serine racemase activity was detected in the membrane but not in the cytoplasmic fraction after centrifugation of sonicated cells, whereas alanine racemase activity was located almost exclusively in the cytoplasm. When the protein was overexpressed as a polypeptide lacking the predicted transmembrane domain, serine racemase activity was detected in the cytoplasm. The serine racemase activity was partially (64%) inhibited by D-cycloserine, whereas host alanine racemase activity was almost totally inhibited (97%). Serine racemase activity was also detected in membrane preparations of constitutively vancomycin-resistant E. gallinarum BM4174 but not in BM4175, in which insertional inactivation of the vanC-1 D-Ala:D-Ser ligase gene probably had a polar effect on expression of the vanXY(c) and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Alr alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Alr were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Alr and that the protein could exist as a dimer. These results indicate that the source of D-serine for peptidoglycan synthesis in vancomycin-resistant enterococci expressing the VanC phenotype involves racemization of L- to D-serine by a membrane-bound serine racemase.