Host-specific enzyme-substrate (ES) interactions in SPM-1 metallo-

GONZÁLEZ LJ; MORENO DM; BONOMO RA; VILA AJ

Denver, Colorado

Congreso; Interscience Conference on Antimicrobial Agents and Chemotherapy 2013; 2013

American Society for Microbiology

Background: Pseudomonas aeruginosa (Pa) is one of the most virulent and resistant pathogens in the clinic. Unfortunately, Pa acquired genes encoding MBLs, Zn(II)-enzymes able to hydrolyze most beta-lactam antibiotics. SPM-1 is an MBL produced only by Pa, while other MBLs are found in different bacteria. Despite similar active sites, the resistance profile of MBLs towards beta-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MBLs in that in that it contains "atypical" second sphere residues (S84, G121) inside the protein. Our goal is to explore the role of these SS residues on catalytic activity and metal uptake in a native context. Methods: The blaSPM-1 gene from Pa 48-1997A was introduced into Pa PAO where codon randomization and selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, western blots, Zn requirements, and protein stability was assessed. Results. Screening revealed 15 resistant variants (10 single, 5 double mutants),suggesting that wild type (WT) residues are not essential for resistance. None of the isolated mutants carried mutations prevalent in MBLs (G121R/H). With few exceptions, mutants conferred lower levels of resistance than WT (MICs differences up to 6 dilutions). Periplasmic activities revealed that WT combination (S84/G121), and mutant G121A, is advantageous only for hydrolyzing latest antipseudomonal beta-lactam antibiotics (cefepime and imipenem) with hydrolysis rates 2.3 to 100-fold higher than the rest of the mutants. Limiting Zn(II) availability affected MICs: WT and G121S were the most resistant to metal deprivation (MIC drop of 1 dilution) while G121A the most sensitive (MIC 64-fold lower). This was correlated with metal stabilization of periplasmic mutants (DeltaTm between holo- and metal-free derivatives), indicating that SS residues modulate Zn(II) binding affinity, which is optimized in WT enzyme. Conclusion. SS residues optimize the resistance profile of SPM-1 by modulating the substrate preference and Zn(II) binding features according to specific requirements in Pa. This work provides an example of molecular evolution to meet the specific needs of a pathogenic bacterial host by means of subtle mutations outside the active site.