Mechanistic Studies on the Mononuclear Zn^II-Containing Metallo-β-lactamase ImiS from Aeromonas sobria

Abstract

In an effort to understand the reaction mechanism of a B2 metallo-β-lactamase, steady-state and presteady state kinetic and rapid-freeze quench EPR studies were conducted on ImiS and its reaction with imipenem and meropenem. pH Dependence studies revealed no inflection points in the pH range of 5.0 -8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer. Sitedirected mutagenesis studies revealed that Lys224 plays a catalytic role in ImiS, while the side chain of Asn233 does not play a role in binding or catalysis. Stopped-flow fluorescence studies on ImiS, which monitor changes in tryptophan fluorescence on the enzyme, and its reaction with imipenem and meropenem revealed biphasic fluorescence time courses with a rate of fluorescence loss of 160 s −1 and a slower rate of fluorescence regain of 98 s −1 . Stopped-flow UV-Vis studies, which monitor the concentration of substrate, revealed a rapid loss in absorbance during catalysis with a rate of 97 s −1 . These results suggest that the rate-limiting step in the reaction catalyzed by ImiS is C-N bond cleavage. Rapid-freeze quench EPR studies on Co(II)-substituted ImiS demonstrated the appearance of a rhombic signal after 10 milliseconds that is assigned to a reaction intermediate that has a 5-coordinate metal center. A distinct product (EP) complex was also observed and began to appear in 18-19 milliseconds. Taken together, these results allow for a reaction mechanism to be offered for the B2 metallo-β-lactamases and demonstrates that the mononuclear Zn(II)-and dinuclear Zn(II)-containing enzymes share a common rate-limiting step, which is C-N bond cleavage.Bacterial resistance to antibiotics is a growing clinical concern (1,2). Zn(II)-containing β-lactamases (metallo-β-lactamases, MβL's) contain 1-2 moles of Zn(II) per mole of enzyme, hydrolyze all known cephalosporins, carbapenems and penicillins, are not inhibited by clavulanic acid and other classical β-lactamase inhibitors, and have no known clinically-useful inhibitor towards them (3,4). Previous studies have shown that there is significant structural and mechanistic diversity among the MβL's, leading to the grouping of the enzymes into three distinct subclasses: B1, B2, and B3 (5,6). Sequence identity ranges from 25-40% between members in one subclass and from 10-20% between members in different subclasses. Subclass B1 enzymes have been found in strains of Bacillus, Bacteroides, Pseudomonas, Serratia, and Chryseobacterium, and subclass B3 enzymes have been found in strains of Stenotrophomonas, Legionella, Fluoribacter, Janthinobacterium and Caulobacter (3,4). Enzymes from the B1 and B3 subclasses have broad substrate profiles and require two Zn(II) † This work was supported by the National Institutes of Health (GM40052 to MWC; AI056231 to BB, and EB001980 to the Medical College of Wisconsin).*To whom correspondence should be addressed: M. W. Crowder, e-mail: crowdemw@muohio.edu, phone: (513) 529-7274, fax: (513) 529-5715. NIH Public Access Author ManuscriptBiochemistry. A...

Common Mechanistic Features among Metallo-β-lactamases

“…The calculated activation free energy for the reaction is in good agreement with experimental findings, and the proposed mechanism is consistent with the studies by Crowder and coworkers (30), who showed that the rate-limiting step is the C-N ␤-lactam cleavage and involves a proton transfer event. The catalytic mechanism is also consistent with previous experimental (52) and theoretical (22) studies, while pointing to the presence of a second water molecule in the active site as crucial element for an efficient ␤-lactam hydrolysis.…”

“…However, this hypothesis is not consistent with experimental studies by Crowder and co-workers (30), who have shown that the reaction catalyzed by the B2 M␤L ImiS from Aeromonas veronii bv. Sobria is characterized by a rate-determining proton transfer step.…”

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

“…In one of them Table S1. involves the Zn2 ion) (16,17,46,48). This suggests that a strategy aimed for the design of a general inhibitor could be pursued by targeting the Zn2 site.…”

“…The issue that remains unsolved is the identification of the nucleophile in Zn2-only enzymes. The absence of the Zn1 site in B2 lactamases has led to the proposal of a non-metal activated nucleophile, a hypothesis later supported by theoretical and enzymological studies (48,49). However, the possibility of a Zn2-bound nucleophile cannot been definitively ruled out.…”

Quantum Mechanics/Molecular Mechanics modeling of biological relevant reactions catalyzed by enzymes

“…[60] Initially, a reduced number of structures of B2 MβL, CphA from Aeromonas hydrophila, [61] ImiS from Aeromonas veronii bv. Sobria [62] and Sfh.I from Serratia fonticola, [60] have been cristalyzed, being the CphA the most studied one. In particular, three different structures, two of them in the apo form and the last one corresponding to the N220G mutant in complex with a biapenem (BIA) derivative, were obtained.…”