Purification, Characterization, and Kinetic Studies of a SolubleBacteroides fragilis Metallo-β-lactamase That Provides Multiple Antibiotic Resistance

Wang, Zhigang; Benkovic, Stephen J.

doi:10.1074/jbc.273.35.22402

Cited by 113 publications

(205 citation statements)

References 38 publications

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“…These spectra are similar to those previously reported for wild-type L1 and nitrocefin (51), and the features can be attributed to substrate decay, product formation, and formation and decay of a ring-opened, nitrogen anionic intermediate, respectively (51,53,54). Under these conditions, the ϳ0.7 M intermediate was formed during the first 10 ms of the reaction (Fig.…”

Section: Resultssupporting

confidence: 88%

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

Garrity

Carenbauer

Herron

et al. 2004

Journal of Biological Chemistry

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Metallo-␤-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a ␤-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 ؎ 0.2, 1.8 ؎ 0.2, and 1.1 ؎ 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10-to 1000-fold drops in k cat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pK a values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.

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Section: Resultssupporting

confidence: 88%

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

Garrity

Carenbauer

Herron

et al. 2004

Journal of Biological Chemistry

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“…The Zn2 site has an AspCys-His triad in the B1 and B2 subclasses, and the Cys residue is substituted by His in the B3 subclass. It is well established that B1 and B3 subclass ␤-lactamases are catalytically active with one zinc cofactor, typically in the Zn1 site (7), and the second zinc typically enhances the catalytic activity (11)(12)(13)(14). However, the second zinc ion inhibits, rather than enhances, the catalytic activity of subclass B2 enzymes (15), which are only found in Aeromonas and have a strong preference toward carbapenems (6).…”

mentioning

confidence: 99%

Catalytic Mechanism of Class B2 Metallo-β-lactamase

Xie

Guo

2006

Journal of Biological Chemistry

105

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The initial nucleophilic substitution step of biapenem hydrolysis catalyzed by a subclass B2 metallo-␤-lactamase (CphA from Aeromonas hydrophila) is investigated using hybrid quantum mechanical/molecular mechanical methods and density functional theory. We focused on a recently proposed catalytic mechanism that involves a non-metal-binding water nucleophile in the active site of the monozinc CphA. Both theoretical models identified a single transition state featuring nearly concomitant nucleophilic addition and elimination steps, and the activation free energy from the potential of mean force calculations was estimated to be ϳ14 kcal/ mol. The theoretical results also identified the general base for activating the water nucleophile to be the metal-binding Asp-120 rather than His-118, as suggested earlier. The protonation of Asp-120 leads to cleavage of the O ␦2 -Zn coordination bond, whereas the negatively charged nitrogen leaving group resulting from the ring opening replaces Asp-120 as the fourth ligand of the sole zinc ion. The electrophilic catalysis by the metal ion provides sufficient stabilization for the leaving group to avoid a tetrahedral intermediate. The theoretical studies provided detailed insights into the catalytic strategy of this unique metallo-␤-lactamase.The excessive use and abuse of ␤-lactam antibiotics have accelerated the spread of drug-resistant bacterial strains. The unprecedented level of antibiotic resistance threatens to destroy their efficacy in treating infectious diseases, posing a grand challenge to public health (1). The primary defense strategy adopted by bacteria is to deactivate the antibiotics by hydrolytic cleavage of the ␤-lactam ring, catalyzed by ␤-lactamases (2). These enzymes can be divided into four classes (3). Enzymes in classes A, C, and D utilize an active site serine in the covalent catalysis of the ␤-lactam hydrolysis, whereas class B consists of metalloenzymes with one or two zinc cofactors. Although the catalytic mechanism of the serine-based enzymes is relatively well established (2), our understanding of class B ␤-lactamases is less developed (4).Metallo-␤-lactamases often have very broad substrate spectra (5) stemming apparently from the metal-dependent catalytic mechanism. Despite much effort, no clinically effective inhibitor has been found. On the other hand, increasing evidence in recent years has pointed to rapid proliferation of these metalloenzymes in pathogenic microorganisms (6). Hence, these enzymes represent a potentially more potent threat to the existing arsenal of ␤-lactam antibiotics than other classes of ␤-lactamases (5, 6).Class B ␤-lactamases can be further separated into three subclasses. Despite considerable sequence diversity (4), their catalytic scaffolds are relatively conserved. All class B ␤-lactamases have two potential metal binding sites (7-11). Protein ligands in the so-called Zn1 site include three His residues in the B1 and B3 subclasses, but a His residue is replaced by Asn in B2 subclass ␤-lactamases. The Zn2 site has an Asp...

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“…The over-expression of MβL CcrA was carried out as previously described (Wang and Benkovic 1998). Briefly, the plasmid pMSZ02 was transformed into BL21 (DE3) E. coli cells, the colonies containing the plasmid were used to inoculate 50 ml LB medium containing 25 µg/mL kanamycin.…”

Section: Over-expression Of Ccramentioning

confidence: 99%

“…However, the widespread use of β-lactam antibiotics has put tremendous selective pressure on bacterial strains to devise the mechanisms to escape the lethal action of the drugs (Wang and Benkovic 1998). The most common and efficient strategy employed by the strains to resist the β-lactam compounds is the expression of β-lactamases (Bebrone 2007).…”

Section: Introductionmentioning

confidence: 99%

“…MβL CcrA used in early biochemical and structural researches has been over-expressed in E. coli as inclusion bodies and the active CcrA is obtained by renaturing the 8 M urea-solubilized inclusion bodies in metal ion-containing buffer (Yang et al 1992;Concha et al 1996;Crowder et al 1996). This preparation process is subject to the aggregation of the enzyme at high protein concentration and is not reliably reproducible (Wang and Benkovic 1998). For this reason, Z. Wang successfully constructed a new overexpression plasmid pMSZ02 for soluble CcrA.…”

Section: Introductionmentioning

confidence: 99%

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Purification of metallo-B-lactamase CcrA from Bacteroides fragilis with salting-out method

Liu

Zhai

Shi

et al. 2013

Braz. arch. biol. technol.

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Purification, Characterization, and Kinetic Studies of a SolubleBacteroides fragilis Metallo-β-lactamase That Provides Multiple Antibiotic Resistance

Cited by 113 publications

References 38 publications

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

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

Catalytic Mechanism of Class B2 Metallo-β-lactamase

Purification of metallo-B-lactamase CcrA from Bacteroides fragilis with salting-out method

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