Characterization of the Metal-Binding Sites of the β-Lactamase from Bacteroides fragilis

Abstract

In an effort to better understand the structure and function of the metallo-beta-lactamase from Bacteroides fragilis, spectroscopic and metal-binding studies were performed on the native, metal-substituted, and mutant forms of the enzyme. Atomic absorption studies demonstrate that the native B. fragilis enzyme tightly binds 2 mol of Zn(II) and, along with mutagenesis studies, that the presence of both metal ions is required for full catalytic activity. EPR spectroscopy was used to confirm that the Co(II)-substituted beta-lactamase binds 2 mol of Co(II) per mole of enzyme, that the two Co(II)'s are highspin and probably uncoupled, with apparent g values of 6.5, 4.2, and 2.0, and that the coordination number of the Co(II) is 5 or 6. This number of ligands for the Co(II)-substituted enzyme is confirmed by UV-Vis spectra, which demonstrate the presence of very weak d-d transitions between 550 and 650 nm (epsilon approximately 30 M-1.cm-1) and an intense feature at 320 nm (epsilon approximately 1570 M-1.cm-1). The latter is assigned to a cysteine sulfur to Co(II) ligand-to-metal charge transfer band, and this assignment is confirmed by the disappearance of this band in the UV-Vis spectrum of a Co(II)-substituted C168S mutant. H NMR studies on the Co(II)-substituted enzyme suggest the presence of three histidine ligands bound to Co(II). Taken together, these studies support the sequence comparison study of Rasmussen et al., in which there is a catalytic metal-binding site with three histidines and one cysteine (C168). The remaining ligands are postulated to be water molecules involved in catalysis. Mutagenesis studies, in combination with activity assays and metal-binding studies, have been used to identify Asp61, Asp90, Asp152, and Asp183 as possible ligands to the second metal-binding site, with Asp90 and Asp152 having a pronounced effect on kcat. These results are discussed in light of the recent crystal structure of the metallo-beta-lactamase from B. cereus.

Spectroscopic studies on Arabidopsis ETHE1, a glyoxalase II-like protein

“…Hydrolysis of benzoylglycyl-L-phenylalanine was monitored at 254 nm [18]. Nitrocefin (Becton-Dickinson) hydrolysis was monitored at 485 nm [19]. Methylglyoxal was assayed colorimetrically by using the 2,3-dinitrophenylhydrazine-alkali reaction [20].…”

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

“…However, through the analysis of recombinant GLX2-2, we demonstrated that glyoxalase II is a Zn(II)-requiring enzyme (18). All species of glyoxalase II, including human, yeast, and Arabidopsis, contain a highly conserved metal binding domain (THXHXDH) that is also present in the family of metallo-␤-lactamases, which are known to require Zn(II) (16,18,20,21). Based on its similarity to the metallo-␤-lactamases, we predicted that glyoxalase II binds two Zn(II) ions, utilizing five histidines, two aspartic acids, and a bridging water molecule (18).…”

The identification of metal‐binding ligand residues in metalloproteins using nuclear magnetic resonance spectroscopy

“…In BCII, kinetic studies indicate that the binding affinity of each zinc ion differ by three orders of magnitude (Baldwin et al, 1978;Sutton et al, 1987), whereas both metals appear to possess similar binding affinities in CcrA (Concha et al, 1996;Crowder et al, 1996). A possible cause for this significant difference in metal affinity may involve the substitution of CyslO4 in CcrA for an arginine in BCII.…”