Arias Cesar A, Weisner Jan, Blackburn Jonathan M, Reynolds Peter E
Department of Biochemistry, University of Cambridge, Tennis Court Road, The Downing Site, Cambridge CB2 1QW, UK1.
Microbiology (Reading). 2000 Jul;146 ( Pt 7):1727-1734. doi: 10.1099/00221287-146-7-1727.
Vancomycin resistance in Enterococcus gallinarum results from the production of UDP-MurNAc-pentapeptide[D-Ser]. VanT, a membrane-bound serine racemase, is one of three proteins essential for this resistance. To investigate the selectivity of racemization of L-Ser or L-Ala by VanT, a strain of Escherichia coli TKL-10 that requires D-Ala for growth at 42 degrees C was used as host for transformation experiments using plasmids containing the full-length vanT from Ent. gallinarum or the alanine racemase gene (alr) of Bacillus stearothermophilus: both plasmids were able to complement E. coli TKL-10 at 42 degrees C. No alanine or serine racemase activities were detected in the host strain E. coli TKL-10 grown at 30, 34 or 37 degrees C. Serine and alanine racemase activities were found almost exclusively (96%) in the membrane fraction of E. coli TKL-10/pCA4(vanT): the alanine racemase activity of VanT was 14% of the serine racemase activity in both E. coli TKL-10/pCA4(vanT) and E. coli XL-1 Blue/pCA4(vanT). Alanine racemase activity was present mainly (95%) in the cytoplasmic fraction of E. coli TKL-10/pJW40(alr), with a trace (1.6%) of serine racemase activity. Additionally, DNA encoding the soluble domain of VanT was cloned and expressed in E. coli M15 as a His-tagged polypeptide and purified: this polypeptide also exhibited both serine and alanine racemase activities; the latter was approximately 18% of the serine racemase activity, similar to that of the full-length, membrane-bound enzyme. N-terminal sequencing of the purified His-tagged polypeptide revealed a single amino acid sequence, indicating that the formation of heterodimers between subunits of His-tagged C-VanT and endogenous alanine racemases from E. coli was unlikely. The authors conclude that the membrane-bound serine racemase VanT also has alanine racemase activity but is able to racemize serine more efficiently than alanine, and that the cytoplasmic domain is responsible for the racemase activity.
鹑鸡肠球菌中的万古霉素耐药性源于UDP - 胞壁酰五肽[D - 丝氨酸]的产生。VanT是一种膜结合丝氨酸消旋酶，是这种耐药性所必需的三种蛋白质之一。为了研究VanT对L - 丝氨酸或L - 丙氨酸消旋作用的选择性，一株在42℃需要D - 丙氨酸才能生长的大肠杆菌TKL - 10菌株被用作宿主，进行转化实验，所用质粒含有来自鹑鸡肠球菌的全长vanT或嗜热脂肪芽孢杆菌的丙氨酸消旋酶基因（alr）：两种质粒都能够在42℃时互补大肠杆菌TKL - 10。在30、34或37℃生长的宿主菌株大肠杆菌TKL - 10中未检测到丙氨酸或丝氨酸消旋酶活性。丝氨酸和丙氨酸消旋酶活性几乎完全（96%）存在于大肠杆菌TKL - 10/pCA4(vanT)的膜组分中：在大肠杆菌TKL - 10/pCA4(vanT)和大肠杆菌XL - 1 Blue/pCA4(vanT)中，VanT的丙氨酸消旋酶活性是丝氨酸消旋酶活性的14%。丙氨酸消旋酶活性主要（95%）存在于大肠杆菌TKL - 10/pJW40(alr)的细胞质组分中，有微量（1.6%）的丝氨酸消旋酶活性。此外，编码VanT可溶性结构域的DNA被克隆并在大肠杆菌M15中作为His标签多肽表达并纯化：该多肽也表现出丝氨酸和丙氨酸消旋酶活性；后者约为丝氨酸消旋酶活性的18%，与全长膜结合酶相似。对纯化的His标签多肽进行N端测序揭示了单一氨基酸序列，表明His标签化的C - VanT亚基与大肠杆菌内源性丙氨酸消旋酶之间形成异源二聚体的可能性不大。作者得出结论，膜结合丝氨酸消旋酶VanT也具有丙氨酸消旋酶活性，但消旋丝氨酸比丙氨酸更有效，并且细胞质结构域负责消旋酶活性。
