On the Mechanism of the Metallo-β-lactamase from Bacteroides fragilis

Abstract

The catalytic mechanism of metallo-beta-lactamase from Bacteroides fragilis, a dinuclear Zn(II)-containing enzyme responsible for multiple antibiotic resistance, has been investigated by using nitrocefin as a substrate. Rapid-scanning and single-wavelength stopped-flow studies revealed the accumulation during turnover of an enzyme-bound intermediate with intense absorbance at 665 nm (epsilon = 30 000 M(-1) cm(-1)). The proposed minimum kinetic mechanism for the B. fragilis metallo-beta-lactamase-catalyzed nitrocefin hydrolysis [Wang, Z., and Benkovic, S. J. (1998) J. Biol. Chem. 273, 22402-22408] was confirmed, and more accurate kinetic parameters were obtained from computer simulations and fitting. The intermediate was shown to be a novel anionic species bound to the enzyme through a Zn-acyl linkage and contains a negatively charged nitrogen leaving group. This is the first time such an intermediate was observed in the catalytic cycle of a Zn(II)-containing hydrolase and is evidence for a unique beta-lactam hydrolysis mechanism in which the amine can leave as an anion; prior protonation is not required. The electrostatic interaction between the negatively charged intermediate and the positively charged dinuclear Zn(II) center of the enzyme is important for stabilization of the intermediate. The catalytic reaction was accelerated in the presence of exogenous nucleophiles or anions, and neither the product nor the enzyme was modified during turnover, indicating that a Zn-bound hydroxide (rather than Asp-103) is the active site nucleophile. On the basis of all the information on hand, a catalytic mechanism of the B. fragilis metallo-beta-lactamase is proposed.

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…This fact highlights its amazing chemical versatility. In the case of M␤Ls, the Zn(II) ion is able to contribute to ␤-lactam hydrolysis by 1) lowering the pK a of a bound water molecule, which may act as a nucleophile, providing a high local concentration of hydroxide ions at neutral pH (37,57); 2) acting as a Lewis acid, polarizing the CϭO bond and therefore augmenting the electrophilic nature of the carbonyl carbon (57); and 3) stabilizing a negative charge in the bridging nitrogen of the lactam moiety, after C-N bond cleavage (16,17,24,36,58,59). These three roles have been invoked for the two metal binding sites in M␤Ls, but it is not clear yet which of them are essential for catalysis.…”

“…Benkovic and co-workers (16,17) reported for the first time the accumulation of a reaction intermediate in the hydrolysis of nitrocefin by the B1 enzyme CcrA from B. fragilis. The same intermediate was also reported for the B3 enzyme L1 (37,46) and for an evolved mutant of the B1 lactamase BcII (39).…”

“…Here we report a series of experiments that allow us to propose that the metal site in GOB is involved in stabilization of an anionic intermediate that accumulates during catalysis, thus favoring C-N bond cleavage after the nucleophilic attack. These results are unprecedented because these intermediates have been only identified in catalysis by dinuclear lactamases (16,17,37,39) and disclose the existence of a common catalytic feature in M␤Ls from all subclasses.…”

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

“…Currently, there are two proposed reaction mechanisms for the dinuclear Zn(II) ␤-lactamases, depending on the substrate. When nitrocefin is used as the substrate, a ring-opened, nitrogen anionic intermediate is thought to form, and the protonation of this nitrogen anion is rate-limiting (51,54,68). When other substrates are used, the breakdown of a tetrahedral intermediate, which occurs via the protonation of the ␤-lactam ring nitrogen, is thought to be rate-limiting (54).…”

X-ray Absorption Spectroscopy of the Zinc-Binding Sites in the Class B2 Metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria

“…Considerable information exists regarding the B1 and B3 enzymes, including X-ray diffraction (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), spectroscopic (9,(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42), mechanistic (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59), and computational studies (60)(61)(62)(63)(64)…”