Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins
Structures of two kinetic intermediates reveal species specificity of penicillin-binding proteins
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DOI：
10.1016/s0022-2836(02)00742-8
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发表时间：
2002-09-06
影响因子：
5.6
通讯作者：
Kelly, JA
中科院分区：
文献类型：
--
作者：
McDonough, MA;Anderson, JW;Kelly, JA
青霉素结合蛋白（PBP）是青霉素和头孢菌素等β-内酰胺类抗生素的靶酶，催化细菌细胞壁生物合成的最后一步肽聚糖交联。β-内酰胺抑制该反应，因为它们模拟细胞壁结构的D-丙氨酰-D-丙氨酸肽前体。先前的晶体学研究已经描述了β-内酰胺结合和抑制的位点，但它们未能显示D-Ala-D-Ala底物的结合。我们在这里提出的第一个高分辨率的晶体结构的PBP，D-Ala-D-Ala-肽酶的链霉菌菌株R61，非共价复合的高度特异性片段（甘氨酰-L-α-氨基-ε-庚二酰-D-Ala-D-Ala）的细胞壁前体在酶底物和酶产物的形式。酶-底物Henri-Michaelis复合物的1.9埃分辨率结构是通过使用失活的酶实现的，所述失活的酶通过交联两个催化重要残基Tyr 159和Lys 65形成。DD-肽酶的未交联的活性形式在1.25埃分辨率下的第二个结构显示了羧肽酶反应的两种产物的非共价结合。两种晶体结构中明确的底物结合位点显示了一个亚位点，该亚位点与细菌物种之间不同的天然细胞壁底物的一部分互补。此外，结构显示了11个水分子从活性位点的置换，负责底物结合的残基的位置，并清楚地证明了Lys 65和/或Tyr 159对于与供体肽的酰化步骤的必要性。本文所述的复合结构与其他已知PBPs的结构的比较表明，物种靶向抗生素的设计作为对抗β-内酰胺酶引起的细菌耐药性的策略。(C)2002爱思唯尔科技有限公司。保留所有权利。
Penicillin-binding proteins (PBPs), the target enzymes of beta-lactam antibiotics such as penicillins and cephalosporins, catalyze the final peptidoglycan cross-linking step of bacterial cell-wall biosynthesis. beta-Lactams inhibit this reaction because they mimic the D-alanyl-D-alanine peptide precursors of cell-wall structure. Prior crystallographic studies have described the site of beta-lactam binding and inhibition, but they have failed to show the binding Of D-Ala-D-Ala substrates. We present here the first high-resolution crystallographic structures of a PBP, D-Ala-D-Ala-peptidase of Streptomyces sp. strain R61, non-covalently complexed with a highly specific fragment (glycyl-L-alpha-amino-epsilon-pimelyl-D-Ala-D-Ala) of the cell-wall precursor in both enzyme-substrate and enzyme-product forms. The 1.9 Angstrom resolution structure of the enzyme-substrate Henri-Michaelis complex was achieved by using inactivated enzyme, which was formed by cross-linking two catalytically important residues Tyr159 and Lys65. The second structure at 1.25 Angstrom resolution of the uncross-linked, active form of the DD-peptidase shows the non-covalent binding of the two products of the carboxypeptidase reaction. The well-defined substrate-binding site in the two crystallographic structures shows a subsite that is complementary to a portion of the natural cell-wall substrate that varies among bacterial species. In addition, the structures show the displacement of 11 water molecules from the active site, the location of residues responsible for substrate binding, and clearly demonstrate the necessity of Lys65 and or Tyr159 for the acylation step with the donor peptide. Comparison of the complexed structures described here with the structures of other known PBPs suggests the design of species-targeted antibiotics as a counter-strategy towards beta-lactamase-elicited bacterial resistance. (C) 2002 Elsevier Science Ltd. All rights reserved.