The basis for resistance to β-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus
The basis for resistance to β-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus
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DOI：
10.1074/jbc.m403589200
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发表时间：
2004-09-24
影响因子：
4.8
通讯作者：
Mobashery, S
中科院分区：
文献类型：
--
作者：
Fuda, C;Suvorov, M;Mobashery, S
金黄色葡萄球菌的青霉素结合蛋白2a（PBP 2a）难以被可用的β-内酰胺抗生素抑制，导致对这些抗生素的耐药性。S.已获得PBP 2a的mecA基因的金黄色葡萄球菌被称为耐甲氧西林金黄色葡萄球菌。金黄色葡萄球菌（MRSA）。将mecA基因克隆到大肠杆菌中进行表达，并纯化PBP 2a。评价了几种β-内酰胺类抗生素（青霉素类、头孢菌素类和碳青霉烯类）与PBP 2a相互作用的动力学参数。该酶以两种方式表现出对β-内酰胺抗生素在活性位点丝氨酸残基处的共价修饰的抗性。首先，酰化的微观速率常数（k（2））比青霉素敏感的青霉素结合蛋白的相应测定值衰减3至4个数量级。第二，该酶显示出与抗生素的非共价预酰化复合物的解离常数（K-d）升高，其形成最终将导致酶酰化。这两个因素协同作用，有效地阻止了抗生素在体内的酶酰化，从而产生耐药性。由于有机会在体外实验中形成酰基酶物种，圆二色性测量显示，酶在该过程中经历了大量的构象变化，这将导致酶酰化。观察到的构象变化可能是这种酶如何在交联细菌细胞壁中发挥催化功能的标志。
Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k(2)) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (K-d) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.