Makena Anne, Brem Jürgen, Pfeffer Inga, Geffen Rebecca E J, Wilkins Sarah E, Tarhonskaya Hanna, Flashman Emily, Phee Lynette M, Wareham David W, Schofield Christopher J
Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
Antimicrobial Research Group, Queen Mary University London, London E1 2AT, UK.
J Antimicrob Chemother. 2015 Feb;70(2):463-9. doi: 10.1093/jac/dku403. Epub 2014 Oct 16.
Metallo-β-lactamase (MBL)-based resistance is a threat to the use of most β-lactam antibiotics. Multiple variants of the New Delhi MBL (NDM) have recently been reported. Previous reports indicate that the substitutions affect NDM activity despite being located outside the active site. This study compares the biochemical properties of seven clinically reported NDM variants.
NDM variants were generated by site-directed mutagenesis; recombinant proteins were purified to near homogeneity. Thermal stability and secondary structures of the variants were investigated using differential scanning fluorimetry and circular dichroism; kinetic parameters and MIC values were investigated for representative carbapenem, cephalosporin and penicillin substrates.
The substitutions did not affect the overall folds of the NDM variants, within limits of detection; however, differences in thermal stabilities were observed. NDM-8 was the most stable variant with a melting temperature of 72°C compared with 60°C for NDM-1. In contrast to some previous studies, kcat/KM values were similar for carbapenem and penicillin substrates for NDM variants, but differences in kinetics were observed for cephalosporin substrates. Apparent substrate inhibition was observed with nitrocefin for variants containing the M154L substitution. In all cases, cefoxitin and ceftazidime were poorly hydrolysed with kcat/KM values <1 s(-1) μM(-1).
These results do not define major differences in the catalytic efficiencies of the studied NDM variants and carbapenem or penicillin substrates. Differences in the kinetics of cephalosporin hydrolysis were observed. The results do reveal that the clinically observed substitutions can make substantial differences in thermodynamic stability, suggesting that this may be a factor in MBL evolution.
基于金属β-内酰胺酶（MBL）的耐药性对大多数β-内酰胺类抗生素的使用构成威胁。最近报道了新德里金属β-内酰胺酶（NDM）的多个变体。先前的报道表明，尽管这些取代位于活性位点之外，但仍会影响NDM的活性。本研究比较了七种临床报道的NDM变体的生化特性。
通过定点诱变产生NDM变体；将重组蛋白纯化至接近均一。使用差示扫描荧光法和圆二色性研究变体的热稳定性和二级结构；针对代表性的碳青霉烯类、头孢菌素类和青霉素类底物研究动力学参数和最低抑菌浓度（MIC）值。
在所检测的限度内，这些取代并未影响NDM变体的整体折叠；然而，观察到了热稳定性的差异。NDM-8是最稳定的变体，其解链温度为72°C，而NDM-1为60°C。与一些先前的研究不同，NDM变体对碳青霉烯类和青霉素类底物的催化常数与米氏常数之比（kcat/KM）值相似，但头孢菌素类底物的动力学存在差异。对于含有M154L取代的变体，用硝噻吩青霉素观察到明显的底物抑制。在所有情况下，头孢西丁和头孢他啶的水解效果较差，kcat/KM值<1 s(-1) μM(-1)。
这些结果并未明确所研究的NDM变体与碳青霉烯类或青霉素类底物在催化效率上的主要差异。观察到头孢菌素水解动力学存在差异。结果确实表明，临床上观察到的取代可在热力学稳定性上产生显著差异，这表明这可能是MBL进化的一个因素。
