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

JAVIER M. GONZÁLEZ AND ALEJANDRO J. VILA

Montevideo-Uruguay

Congreso; 6th International Conference of Biological Physics ICBP 2007, 5th Southern Cone Biophysics Congress and 34th Annual Meeting of the Argentinean Biophysical Society; 2007

Sociedad Argentina de Biofisica

Metallo-beta-lactamases (MBLs hereafter) are bacterial binuclear zinc hydrolases that confer broad-spectrum resistance to beta-lactam antibiotics. MBLs require one or two Zn(II) ions for activity and have been classified into three subclasses based on primary sequence homology. Because of their plasmid-borne nature, most of subclass B1 MBLs stand as rapidly evolving resistance mechanisms among pathogenic bacteria found in intensive care units. MBLs belonging to subclass B3 are chromosomal enzymes exhibiting a higher sequence divergence and, presumably, dissimilar catalytic mechanisms. The enzyme BcII from B. cereus is a subclass B1 chromosomal enzyme, strictly related to plasmid-borne MBLs and a wealth of information about it is currently available. Recently, we generated mutants of BcII that resemble the primary zinc coordination 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 most spectroscopic techniques, we decided to employ the Co(II)-substituted forms of both mutants as surrogates of  the corresponding Zn(II) forms. We show that Co(II)-substituted mutants display beta-lactamase activity comparable to the corresponding Zn(II) forms. Stepwise titration of apo-proteins with Co(II) at pH 6 and pH 7.5 showed distinctive features in the UV-Vis region, which allowed us to determine the Co(II) dissociation constants governing the binding process. Such features were in line with the 1H-NMR spectra recorded for each mutant under the same conditions. We conclude that the Co(II) binding mode is analogous to the Zn(II) binding mode, as judged by comparison with previously characterized Zn(II)-dependence of activity profiles of both mutants, thereby making Co(II) substitution a suitable approach to analyze the structure-function relationship in the Zn(II) forms.