Abstract
KPC enzymes are the most common class A carbapenemases globally diffused. The peculiarity of this family of β-lactamases is represented by their ability to hydrolyse all classes of β-lactams, including carbapenems, posing a serious problem to public health. In the present study, seven laboratory mutants of KPC-3 (D228S, D228W, D228M, D228K, D228L, D228I and D228G) were generated by site-saturation mutagenesis to explore the role of residue 228, a non-active site residue. Compared to KPC-3, the seven mutants showed evident differences in kcat and Km values calculated for some penicillins, cephalosporins and carbapenems. In particular, D228S and D228M showed a significant increase of Km values for cefotaxime and ceftazidime. Circular dichroism (CD) experiments have demonstrated that substitution at position 228 does not affect the secondary structure of the mutants. Molecular dynamics (MD) simulations were performed on KPC-3, D228S and D228M uncomplexed and complexed with cefotaxime (substrate). Although the residue 228 is located far from the active site, between α11 helix and β7 sheet in the opposite site of the Ω-loop, amino acid substitution at this position generates mechanical effects in the active site resulting in enzyme activity changes.
Highlights
Introduction βLactamases are bacterial enzymes that efficiently hydrolyse β-lactam antibiotics, the most common antimicrobials used in clinical therapy [1]
To overproduce KPC-3 and D228X enzymes, the blaKPC-3, blaD228S, blaD228W, blaD228M, blaD228K, blaD228L, blaD228I and blaD228G were sub-cloned into the pET-24a(+) vector and inserted by transformation in E. coli BL21(DE3)
The kinetic characterization was performed on KPC-3 and on the following laboratory variants: D228S, D228W, D228K, D228M, D228L, D228I and D228G
Summary
Lactamases are bacterial enzymes that efficiently hydrolyse β-lactam antibiotics, the most common antimicrobials used in clinical therapy [1]. On the basis of catalytic mechanism, β-lactamases include two distinct groups: serine β-lactamases (SBLs), which hydrolyse β-lactams via a catalytic serine, and metallo-β-lactamases (MBLs), which need one or two essential zinc ions for catalysis [2,3]. According to Ambler classification, β-lactamases are categorized into four molecular classes (A, B, C and D) based on primary amino acid similarities [4]. About 3000 different β-lactamases have been identified in Catalysts 2020, 10, 1474; doi:10.3390/catal10121474 www.mdpi.com/journal/catalysts. The class B zinc-dependent β-lactamases are metallo-enzymes which are able to hydrolyse the amide bond of β-lactam ring of most β-lactams, especially carbapenems. On the basis of sequence homologies, three distinct MBL superfamilies have been identified: subclasses
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