Kuzin A P, Sun T, Jorczak-Baillass J, Healy V L, Walsh C T, Knox J R
Department of Molecular and Cell Biology, The University of Connecticut, Storrs, CT 06269-3125, USA.
Structure. 2000 May 15;8(5):463-70. doi: 10.1016/s0969-2126(00)00129-5.
The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins). Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands. The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics. Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase. Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine.
The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution. Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis. Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2). A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2.
Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.
细菌细胞壁及其合成酶是糖肽类抗生素（万古霉素和替考拉宁）和β-内酰胺类抗生素（青霉素和头孢菌素）的作用靶点。细胞壁肽聚糖的生物合成需要相邻聚糖链上的肽基部分进行交联。催化这种交联的D-丙氨酸-D-丙氨酸转肽酶是β-内酰胺类抗生素的作用靶点。相比之下，糖肽类抗生素并不抑制酶的活性，而是直接与D-丙氨酸-D-丙氨酸结合，阻止转肽酶随后的交联反应。肠球菌病原体对万古霉素的临床耐药性已追溯到连接酶的改变，这种改变导致产生D-丙氨酸-D-乳酸而非D-丙氨酸-D-丙氨酸。
通过多波长反常散射（MAD）相位法确定了D-丙氨酸-D-乳酸连接酶的结构，分辨率为2.4埃。嗜热栖热放线菌LmDdl2连接酶与ATP和二甲基次膦酸盐共结晶，产生了ADP和细胞壁肽聚糖生物合成反应中间体的次膦酸酯类似物。将这种D-丙氨酸-D-乳酸连接酶与已知结构的DdlB D-丙氨酸-D-丙氨酸连接酶（一种不产生万古霉素耐药性的野生型酶）进行比较，发现D-乳酸结合位点（亚位点2）的大小和疏水性发生了改变。连接酶与进入亚位点2的底物分子形成氢键的能力有所下降。
D-丙氨酸-D-乳酸连接酶亚位点2的结构差异有助于解释底物特异性的转变（从D-丙氨酸到D-乳酸），这种转变导致革兰氏阳性病原体的细胞壁肽聚糖重塑和万古霉素耐药性。
