Biochemical and structural characterization of CRH-1, an environmental class A carbapenemase from Chromobacterium haemolyticum (Póster)

FLORENCIA BRUNETTI; BÁRBARA GHIGLIONE; DEREJE GUDETA; LUCA GUARDABASSI; GABRIEL GUTKIND; SEBASTIÁN KLINKE; PABLO POWER

Washington DC

Congreso; ASM Microbe 2022; 2022

American Society for Microbiology (ASM)

Background: CRH-1 is a class A beta-lactamase (BLA) isolated from Chromobacterium haemolitycum, a species that is mostly recovered from aquatic environments. This enzyme has 69% amino acid identity with KPC-2, and was proposed as a potential evolutionary ancestor of this carbapenemase. The aim of this study is to explore this potential evolutionary pathway by comparing kinetic parameters, and protein structure obtained from X-ray crystallography, for CRH-1 and KPC-2. Methods: bla genes were cloned in both pMBLe (MIC determination according to CLSI), and pET24a (protein expression and purification). Steady-state kinetic parameters (Km, kcat, kcat/Km) for beta-lactams were determined. Crystals of apo CRH-1 and its complex with avibactam (AVI) were obtained by the hanging drop vapor diffusion method. X-ray diffraction was measured at the Soleil synchrotron (France). Data process was performed with XDS, and the structure was solved by molecular replacement with Phenix. Results: MIC values for most beta-lactams tested were higher in the recombinant clone expressing CRH-1 than in the clone expressing KPC-2, except for aztreonam and the combination ceftazidime/avibactam. MICs for imipenem and meropenem were 8 fold higher for CRH-1. These data correlated with the kinetic constants for carbapenems, hydrolytic efficiencies (kcat/Km) for imipenem and meropenem were 1.6 and 1.4-fold higher respectively, compared to KPC-2 (at expenses of a lower Km in both cases). CRH-1 also exhibited higher kcat/Km for ampicillin, piperacillin and cephalothin. Structures were obtained at high resolution (CRH-1: 1.1 Å; CRH-1/AVI: 1.4 Å). Compared to KPC-2, the class A enzyme (69% id), CRH-1 showed equivalent organization of the active sites as well as similar interactions with AVI. Conclusions: Our results support the hypothesis that some species belonging to the genus Chromobacterium could be potential reservoirs of newer class A carbapenemases which share biochemical and structural features with the globally disseminated carbapenemase KPC-2. The favorable interactions observed with AVI foresees an optimistic scenario of treatment in case this type of enzyme eventually emerges among clinical pathogens in the near future.