Analysis of a mutational hotspot in metallo-beta-lactamases

GUILLERMO BAHR; MARÍA ROCÍO MEINI; ALEJANDRO J. VILA

San Javier, Tucumán

Congreso; SAB 2012: XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

Metallo-beta-lactamases (MBLs) constitute the latest resistance mechanism of bacteria against beta-lactam antibiotics. MBLs require Zn(II) bound to their active sites and are able to hydrolyze all classes of beta-lactams. They are spreading rapidly and new evolved variants are continually discovered while there is no therapeutic inhibitor available for them. Directed Molecular Evolution (DME) strategies simulate the natural evolutionary process in the laboratory. By using DME, we have generated evolved variants of the metallo-beta-lactamase BcII from Bacillus cereus. Four point mutations were responsible for enzyme evolution: G262S and N70S, located below the active site floor and V112A and L250S, located far from it. Hydrolytic efficiency is first optimized by the G262S mutation, but at the cost of losing Zn(II) affinity and stability. The other mutations reconstitute the affinity for the cofactor Zn(II) and stability. Despite the low sequence identity of MBLs, their fold is preserved, and these four mutations appear at regions well conserved among the MBLs. Our aim is to extrapolate the results obtained with BcII to MBLs of high clinical relevance. For this we employed the VIM-2 MBL, an integron-borne lactamase that has been found carried by various opportunistic pathogens in clinical samples worldwide, and which presents 32% sequence identity to BcII. The point mutations found on the DME experiment were replicated on VIM-2. Preliminary results indicate that G262S also has an effect on Zn(II) affinity in the case of VIM-2, showing that DME strategies are very powerful to highlight mutational hotspots that are critical for protein function and thus to provide information to anticipate the evolution of MBLs.