Mutations in the active site of penicillin-binding protein PBP2x from Streptococcus pneumoniae -: Role in the specificity for β-lactam antibiotics
Mutations in the active site of penicillin-binding protein PBP2x from Streptococcus pneumoniae -: Role in the specificity for β-lactam antibiotics
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DOI：
10.1074/jbc.274.27.19175
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发表时间：
1999-07-02
影响因子：
4.8
通讯作者：
Vernet, T
中科院分区：
文献类型：
--
作者：
Mouz, N;Di Guilmi, AM;Vernet, T
从临床耐β-内酰胺类肺炎链球菌分离的青霉素结合蛋白2x(PBP2x)对β-内酰胺类抗生素的亲和力降低。与β-内酰胺敏感链球菌(S-PBP2x)的同源序列相比，它们的转肽酶结构域存在大量的替换，R-PBP2x的序列比较表明突变Gln(552)--&Gt；Glu在耐药发展中起重要作用。在实验室筛选出的头孢菌素突变体经常含有突变Thr(550)-->；Ala，S-PBP2x*与头孢呋辛之间的高分辨结构表明，Gln(552)和Thr(550)属于β3链，与头孢菌素有直接接触。我们研究了在大肠杆菌中表达的可溶性S-PBP2x(S-PBP2x*)第552位和550位的改变对其的影响。与S-PBP2x*相比，突变Q552E降低了青霉素G和头孢噻肟的酰化效率。我们认为，在β3链中引入负电荷与β-内酰胺的负电荷相冲突。突变T550A降低了该蛋白对头孢噻肟的酰化效率，但对青霉素G没有影响，体外数据与这些突变在体内介导的不同耐药性相一致，并强调了它们作为强大的耐药性决定因素的作用。
Penicillin-binding protein 2x (PBP2x) isolated from clinical beta-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for beta-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from beta-lactam-sensitive streptococci (S-PBP2x), Comparison of R-PBP2x sequences suggested that the mutation Gln(552) --> Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr(550) --> Ala, The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln(552) and Thr(550), which belong to strand beta 3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand beta 3 conflicts with the negative charge of the beta-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G, The in vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.