van Alen Ilona, Chikunova Aleksandra, van Zanten Danny B, de Block Amber A, Timmer Monika, Brünle Steffen, Ubbink Marcellus
Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
FEBS J. 2023 Oct;290(20):4933-4949. doi: 10.1111/febs.16892. Epub 2023 Jul 2.
Conserved residues are often considered essential for function, and substitutions in such residues are expected to have a negative influence on the properties of a protein. However, mutations in a few highly conserved residues of the β-lactamase from Mycobacterium tuberculosis, BlaC, were shown to have no or only limited negative effect on the enzyme. One such mutant, D179N, even conveyed increased ceftazidime resistance upon bacterial cells, while displaying good activity against penicillins. The crystal structures of BlaC D179N in resting state and in complex with sulbactam reveal subtle structural changes in the Ω-loop as compared to the structure of wild-type BlaC. Introducing this mutation in four other β-lactamases, CTX-M-14, KPC-2, NMC-A and TEM-1, resulted in decreased antibiotic resistance for penicillins and meropenem. The results demonstrate that the Asp in position 179 is generally essential for class A β-lactamases but not for BlaC, which can be explained by the importance of the interaction with the side chain of Arg164 that is absent in BlaC. It is concluded that Asp179 though conserved is not essential in BlaC, as a consequence of epistasis.
保守残基通常被认为对功能至关重要,预计这些残基的取代会对蛋白质的性质产生负面影响。然而,已证明结核分枝杆菌β-内酰胺酶BlaC的一些高度保守残基发生突变对该酶没有或只有有限的负面影响。其中一个这样的突变体D179N,甚至使细菌细胞对头孢他啶的耐药性增加,同时对青霉素显示出良好的活性。静止状态下和与舒巴坦结合时BlaC D179N的晶体结构显示,与野生型BlaC的结构相比,Ω环存在细微的结构变化。在其他四种β-内酰胺酶CTX-M-14、KPC-2、NMC-A和TEM-1中引入此突变,导致对青霉素和美罗培南的抗生素耐药性降低。结果表明,179位的天冬氨酸对A类β-内酰胺酶通常至关重要,但对BlaC并非如此,这可以通过与BlaC中不存在的Arg164侧链相互作用的重要性来解释。得出的结论是,由于上位性,Asp179虽然保守,但在BlaC中并非必不可少。