The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, UiT The Arctic University of Norway, Tromsø, Norway.
Department of Pharmacy, UiT The Arctic University of Norway, Tromsø, Norway.
J Antimicrob Chemother. 2020 Sep 1;75(9):2554-2563. doi: 10.1093/jac/dkaa175.
MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure-activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure.
To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1.
The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1).
Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity.
These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.
MBLs 形成了一个庞大而多样的细菌酶群体，赋予了对β-内酰胺类抗生素（包括碳青霉烯类）的耐药性。已经确定了 MBLs 的大型环境储库，它们可以作为转移到人类病原体中的来源。因此，对环境和临床罕见的 MBLs 的结构研究可以深入了解结构-活性关系，探索催化和第二壳层残基的作用，这些残基受到选择压力的影响。
研究环境亚型 B1 MBLs MYO-1、SHD-1 和 ECV-1 的结构和活性。
将这些 MBLs 的相应基因克隆到载体中，并在大肠杆菌中表达。对纯化酶的催化效率（kcat/Km）进行了特征描述。测定了不同β-内酰胺类药物（包括青霉素类、头孢菌素类和碳青霉烯类）的酶活性和 MIC。通过 X 射线晶体学（MYO-1 和 ECV-1）或同源建模（SHD-1）来测量热稳定性并解析结构。
在大肠杆菌中表达环境 MBLs 导致了特征性的 MBL 谱，不影响氨曲南的敏感性，并降低了对碳青霉烯类、头孢菌素类和青霉素类的敏感性。与广泛分布的 NDM-1 相比，纯化酶的催化活性差异很大，在 5%至 70%之间。ECV-1 和 SHD-1 的热稳定性比 MYO-1 和 NDM-1 高 8°C。通过使用已解决的结构和分子建模，我们确定了它们第二壳层组成的差异，这可能是它们相对较低的水解活性的原因。
这些结果表明，环境物种作为 MBL 编码基因的储库的重要性。
