Evidence for a dinuclear active site in the metallo-beta-lactamase BcII with substoichiometric Co(II). A new model for metal uptake.
Metallo-beta-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to beta-lactam antibiotics. Metallo-beta-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-beta-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His(116), His(118), and His(196)) and DCH (Asp(120), Cys(221), and His(263)) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-beta-lactamase turnover.
Llarrull
Leticia I
LI
Molecular Biology Division, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina.
Tioni
Mariana F
MF
Kowalski
Jason
J
Bennett
Brian
B
Vila
Alejandro J
AJ
D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D017136:Ion Transport; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D008667:Metalloproteins; D011994:Recombinant Proteins; D003035:Cobalt; C455979:beta-lactamase BcII; D001618:beta-Lactamases; D015032:Zinc