ROLE OF CONFORMATIONAL FLEXIBILITY IN THE FITNESS OF β- LACTAMASES ENZIMES

ALEJANDRO VILA; MARIANO GONZALEZ; MARIA AGUSTINA ROSSI

Santa Fe

Workshop; III Workshop on Magnetic Resonance ?NMR and EPR at the Forefront of Research; 2016

Alternate structural conformers can mediate alternate folds and functions, this diversityis the foundation of ?protein evolvability?, the ability of proteins to rapidly adopt newfunctions within existing folds or even adopt entirely new folds.1,2.Antibiotic resistance mediated by β-lactamases represents an evolutionary paradigm inwhich organismal fitness depends on the catalytic efficiency of a single enzyme, whichcan be divided in two large families: serin-β-lactamases (SBLs) and metallo-β-lactamases (MBLs).3,4 CTX-M enzymes belong to the group of ESBLs (Extendedspectrum β-lactamases). In the classic ESBLs, the increased hydrolytic spectrum camewith enlargement of the active site.5 Instead, CTX -M expanded activity appears to berelated, not with an increase in the active site volume, but with an increased flexibility.This increased flexibility is correlated with higher ceftazidimase activity and lowerstability. Structurally, this can be reconciled with the substitutions D240G and V231A,which occur at either end of the B3 strand. 6,7To test this hypothesis, we will study the dynamics of the polypeptide backbone ofnatural variants of CTX-M-14: CTX-M-9 (V231A), CTX-M-27 (D240G) y CTX-M-16(double mutant) by NMR spectrosopy. It has been reported that the D240G substitutionleads to reduced catalytic efficiency against cefotaxime, the natural substrate, butimproves activity against bulky substrates. The V231A mutation does not affectenzyme activity but, in conjunction with D240G substitution, results in an increaseactivity against the natural substrate and the ceftazidime, the bulkier one.We cloned CTX-M-14 in a high expression plasmid and we generated, by site-directedmutagenesis, its natural variants: CTX-M-9, CTX-M-27 and CTX-M-16. CTX-M-14 waspurified upon homogeneity in minimal medium M9, with a yield of 200 mg/L of protein.The activity of this enzyme was tested against different antibiotics: cefotaxime,cephalothin and ceftazidime, condition in which no hydrolysis was observed.Finally, the HSQC of CTX-M-14 was obtained, according with the signal dispersion ofthe spectrum the protein was properly folded, also it was stable during all time of theassignation experiments.