Structural and functional insights into the substrate specificity of OXA-48-like carbapenemases

Abstract : Antimicrobial resistance is the most alarming emerging problem in infectious diseases. b-Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotics used to treat bacterial infections. However, their utility is being threatened by the worldwide proliferation of b-lactamases (BLs). BL-mediated resistance does not spare even most powerful b-lactams, carbapenems, whose activity is challenged by carbapenemases. OXA-48, a carbapenem-hydrolyzing class D b-lactamase (CHDL) initially identified from a Klebsiella pneumoniae isolate from Turkey in 2001, has since spread globally with the isolation of more than 30 variants. Most OXA-48-like enzymes hydrolyze penicillins at high level, carbapenems at low level and lack significant expanded-spectrum cephalosporin (3GC) hydrolysis, others such as OXA-163 hydrolyze expanded-spectrum cephalosporins and poorly carbapenems. Comparison of OXA-48 tertiary structure with those of other CHDLs revealed small differences located mainly in the loops connecting secondary structure elements, which may vary in length and orientation. The loop located between the b5 and b6 strands (Tyr211 to Pro217) has been suggested to play a major role in carbapenem hydrolysis.To better understand the contribution of the b5-b6 loop in the carbapenem hydrolysis of OXA-48-like carbapenemases, we investigated, using biochemistry and structural biology, natural OXA-48 variants with changes in different loops, replaced each AA of the loop b5-b6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA-18, a clavulanic acid inhibited class D b-lactamase that presents activity against expanded-spectrum cephalosporins and none against carbapenems. The resulting OXA-48loop18 was able to hydrolyze expanded-spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an opening of the active site, being now accessible to b-lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements in the b5-b6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by changes in kcat. By increasing deletions in the b5-b6 loop, starting from Tyr211 to Pro217 and from the Pro217 to Tyr211, the activity against carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revealed the highest 3GC activity, except for one single AA mutant, OXA-48∆P217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized b-lactams to enter the active site. Moreover, the characterization of three novel natural OXA-48 variants revealed structural features important in the observed hydrolysis profile. Thus, the I215-E216 deletion and R214K substitution in the b5-b6 loop of OXA-517 induced the hydrolysis of carbapenems and C3G at high level. In OXA-519, the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the b5-b6 loop, and therefore overall higher Km values were observed compared to OXA-48. The bulkier side chain of L120 in OXA-519 hampers the approach of b-lactam substrate, resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally-encoded OXA-535 that is more distantly related to OXA-48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA-535 presented 98.9% of AA identity with the plasmid-mediated OXA-436 suggesting that the blaOXA-535 gene might be the progenitor of the plasmid-encoded blaOXA-436 gene.Taken together, our work illustrates the propensity of OXA-48 to evolve through mutations to accommodate different substrates in its active site and how the b5–b6 loop determines the specificity of the enzyme.
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Maria Laura Belen Dabos. Structural and functional insights into the substrate specificity of OXA-48-like carbapenemases. Bacteriology. Université Paris-Saclay, 2018. English. ⟨NNT : 2018SACLS402⟩. ⟨tel-02169206v2⟩

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