Raczynska Joanna E, Imiolczyk Barbara, Komorowska Marlena, Sliwiak Joanna, Czyrko-Horczak Justyna, Brzezinski Krzysztof, Jaskolski Mariusz
Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland; Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland.
Int J Biol Macromol. 2020 Apr 28;158:104-115. doi: 10.1016/j.ijbiomac.2020.04.219.
Two accessory loop regions that are present in numerous variants of New Delhi metallo-β-lactamases (NDM) are important for the enzymatic activity. The first one is a flexible loop L3 that is located near the active site and is thought to play an important role in the catalytic process. The second region, Ω loop is located close to a structural element that coordinates two essential zinc ions. Both loops are not involved in any specific interactions with a substrate. Herein, we investigated how the length and hydrophobicity of loop L3 influence the enzymatic activity of NDMs, by analyzing mutants of NDM-1 with various deletions/point mutations within the L3 loop. We also investigated NDM variants with sequence variations/artificial deletions within the Ω loop. For all these variants we determined kinetic parameters for the hydrolysis of ampicillin, imipenem, and a chromogenic cephalosporin (CENTA). None of the mutations in the L3 loop completely abolished the enzymatic activity of NDM-1. Our results suggest that various elements of the loop play different roles in the hydrolysis of different substrates and the flexibility of the loop seems necessary to fulfill the requirements imposed by various substrates. Deletions within the Ω loop usually enhanced the enzymatic activity, particularly for the hydrolysis of ampicillin and imipenem. However, the exact role of the Ω loop in the catalytic reaction remains unclear. In our kinetic tests, the NDM enzymes were inhibited in the β-lactamase reaction by the CENTA substrate. We also present the X-ray crystal structures of the NDM-1, NDM-9 and NDM-12 proteins.
新德里金属β-内酰胺酶（NDM）众多变体中存在的两个辅助环区域对酶活性很重要。第一个是柔性环L3，它位于活性位点附近，被认为在催化过程中起重要作用。第二个区域，Ω环靠近一个协调两个必需锌离子的结构元件。这两个环均不参与与底物的任何特异性相互作用。在此，我们通过分析L3环内具有各种缺失/点突变的NDM-1突变体，研究了环L3的长度和疏水性如何影响NDM的酶活性。我们还研究了Ω环内具有序列变异/人工缺失的NDM变体。对于所有这些变体，我们测定了氨苄西林、亚胺培南和一种显色头孢菌素（CENTA）水解的动力学参数。L3环中的突变均未完全消除NDM-1的酶活性。我们的结果表明，环的各种元件在不同底物的水解中发挥不同作用，并且环的灵活性似乎是满足各种底物所施加要求所必需的。Ω环内的缺失通常会增强酶活性，特别是对于氨苄西林和亚胺培南的水解。然而，Ω环在催化反应中的确切作用仍不清楚。在我们的动力学测试中，CENTA底物在β-内酰胺酶反应中抑制了NDM酶。我们还展示了NDM-1、NDM-9和NDM-12蛋白的X射线晶体结构。
