Spectroscopic analysis of two double mutants of Bacillus cereus Zn(II) beta-lactamase through Co(II) substitution

JAVIER MARCELO GONZALEZ; ALEJANDRO J. VILA

Montevideo

Congreso; 6th International Conference of Biological Physics; 2007

Sociedad Argentina de Biofísica

Metallo-beta-lactamases(MBLs hereafter) are bacterial binuclear zinc hydrolases that confer broad-spectrumresistance to beta-lactam antibiotics. MBLs require one or two Zn(II) ions foractivity and have been classified into three subclasses based on primarysequence homology. Because of their plasmid-borne nature, most of subclass B1MBLs stand as rapidly evolving resistance mechanisms among pathogenic bacteriafound in intensive care units. MBLs belonging to subclass B3 are chromosomalenzymes exhibiting a higher sequence divergence and, presumably, dissimilarcatalytic mechanisms. The enzyme BcII from B.cereus is a subclass B1 chromosomal enzyme, strictly related toplasmid-borne MBLs and a wealth of information about it is currently available.Recently, we generated mutants of BcII that resemble the primary zinccoordination shell located at the active site of B3-MBL L1, and glyoxalase II,a thiolesterase structurally related to MBLs; namely BcII-HS (BcII-R121H/C221S)and BcII-HD (BcII-R121H/C221D).   Since Zn(II) is silent for mostspectroscopic techniques, we decided to employ the Co(II)-substituted forms ofboth mutants as surrogates of  thecorresponding Zn(II) forms. We show that Co(II)-substituted mutants displaybeta-lactamase activity comparable to the corresponding Zn(II) forms. Stepwisetitration of apo-proteins with Co(II) at pH 6 and pH 7.5 showed distinctivefeatures in the UV-Vis region, which allowed us to determine the Co(II)dissociation constants governing the binding process. Such features were inline with the 1H-NMR spectra recorded for each mutant under the sameconditions. We conclude that the Co(II) binding mode is analogous to the Zn(II)binding mode, as judged by comparison with previously characterizedZn(II)-dependence of activity profiles of both mutants, thereby making Co(II)substitution a suitable approach to analyze the structure-function relationshipin the Zn(II) forms.Thiswork has been supported by grants from CONICET, ANPCyT and HHMI to A. J. V.