\A distal mutation from the active site drastically increase Zn(II) affinity of the carbapenemase NDM

DELMONTI, JULIANA.; VILA, ALEJANDRO J. ; MORENO, DIEGO M.

Conferencia; #LatinXChem Twitter Conference 2020; 2020

LatinxChem

NewDelhi Metallo-ß-lactamase (NDM) is a periplasmic Zn(II)-dependent ß-lactamasethat hydrolyzes carbapenems, last resort ß-lactam antibiotics used againstmultiresistant pathogenic bacteria. During infection, the immune response from host withholds nutrient metalions from microbial pathogens. In metal limitation conditions,metallo-ß-lactamases (MBLs), and in particular NDM, lose activity bydissociation of the Zn(II) cofactor. Up to date 29 NDM natural variants werereported, with the substitution M154L being the most frequent. Studies in ourgroup have shown that the tolerance to Zn(II) starvation is selected during theevolution of NDM.Ourresults shown the mutation M154L doesnot improve NDM activity nor stability. However, the substitution M154L alone or in combination with A223V significant impact on the Zn(II) affinityof the protein suggesting that there is a strong evolutionary pressure.Todetermining structuralbases of this huge change, and since crystals structures of metaled NDM-1 andNDM-4 (M154L) were almost superimposable, weperformed NMR studies. Apo NDM-41H-15N- HSQC reflect greater structural flexibility than NDM-1. Thishypothesis was confirmed by classical molecular dynamics that shown theM154L substitution in the non-metalated form causes a greater fluctuation ofthe metal ligands in Loop7.Our results suggest the natural substitution M154Lis selected by strong evolution pressure on NDM favoring the increase of theZn(II) affinity. This biochemical change seen to correlate with an increasingflexibility on the active site.