Arias C A, Martín-Martinez M, Blundell T L, Arthur M, Courvalin P, Reynolds P E
Department of Biochemistry, University of Cambridge, UK.
Mol Microbiol. 1999 Mar;31(6):1653-64. doi: 10.1046/j.1365-2958.1999.01294.x.
Sequence determination of a region downstream from the vanXYc 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 vanXYc and vanT genes. Comparative modelling of the deduced C-terminal domain was based on the alignment of VanT with the Air alanine racemase from Bacillus stearothermophilus. The model revealed that almost all critical amino acids in the active site of Air were conserved in VanT, indicating that the C-terminal domain of VanT is likely to adopt a three-dimensional structure similar to that of Air 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.
鸡肠球菌BM4174中vanXYc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，它编码一个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。该蛋白质在C末端结构域含有一个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。该蛋白质在大肠杆菌中过表达，超声破碎细胞离心后，在膜中检测到丝氨酸消旋酶活性，但在细胞质部分未检测到，而丙氨酸消旋酶活性几乎完全位于细胞质中。当该蛋白质作为缺乏预测跨膜结构域的多肽过表达时，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的鸡肠球菌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vanXYc和vanT基因的表达产生极性影响。基于VanT与嗜热脂肪芽孢杆菌的Air丙氨酸消旋酶的比对，对推导的C末端结构域进行了比较建模。该模型显示，Air活性位点中几乎所有关键氨基酸在VanT中都是保守的，这表明VanT的C末端结构域可能采用与Air相似的三维结构，并且该蛋白质可能以二聚体形式存在。这些结果表明，表达VanC表型的耐万古霉素肠球菌中肽聚糖合成的D-丝氨酸来源涉及由膜结合丝氨酸消旋酶将L-丝氨酸消旋为D-丝氨酸。
