Clavulanic acid is a widely used -lactamase inhibitor whose key -lactam core is formed by -lactam synthetase. -Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by -lactam formation. 32 PP i -ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 ؎ 0.04), it was concluded that -lactam synthesis contributes at least partially to k cat . Site-specific mutation of Lys-443 identified this residue as the ionizable group at pK a ϳ 8.1 apparent in the pH-k cat profile that stabilizes the -lactamforming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on k cat attenuating the observed solvent isotope effect on -lactam formation. Adherence to Kramers' theory gave a slope of 1.66 ؎ 0.08 from a plot of log( o k cat /k cat ) versus log(/ o ) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of >1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 ؎ 0.15) on k cat /K m . The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (⌬H ‡ ϳ 20 kcal/mol) comparable to the chemical barrier of -lactam formation.Resistance to antibiotics is a consequence of their widespread use in the treatment of disease and can arise with dispiriting rapidity after the introduction of a new drug. -Lactam antibiotics constitute 55% of all antibiotics sold despite their prominent use for more than 50 years (1). Their longevity is because of semi-synthetic structural modifications, the discovery of new members of the family from natural sources, and the growing importance of inhibitors of the principal means of resistance, the -lactamases (2). Clavulanic acid is a potent serine -lactamase inhibitor isolated from Streptomyces clavuligerus (3) whose combination with broad spectrum -lactam antibiotics has proven to be highly effective against resistant infections in humans (4, 5). Inhibitor-resistant -lactamases are emerging (1), however, and the need for new entities clinically useful against them is growing.Clavulanic acid is one of only three -lactamase inhibitors in clinical use (Scheme 1) (6). Although other clavams produced in nature may possess antibacterial and antifungal properties, clavulanic acid is the only known clavam with potent -lactamase inhibitory activity owing in part to its 3R,5R stereochemistry (7). It is therefore important to understand the steps involved in this pathway to enable the production of derivatives. Substitution at C-6 of clavulanic acid derivatives, for example, led to broader spectrum inhibition toward ...
Desferrithiocin shows, in comparison with other iron chelators, a similar but lower tendency to bind iron(ii1) under physiological conditions although its biological efficiency in animal models is quite promising.
Abstractβ-Lactam-synthesizing enzymes carbapenam synthetase (CPS) and β-lactam synthetase (β-LS) are evolutionarily linked to a common ancestor, asparagine synthetase B (AS-B). These three relatives catalyze substrate acyl-adenylation and nucleophilic acyl substitution by either an external (AS-B) or internal (CPS, β-LS) nitrogen source. Unlike AS-B, crystal structures of CPS and β-LS revealed a putative Tyr-Glu dyad (CPS, Y345/E380; β-LS, Y348/E382) proposed to deprotonate the respective internal nucleophile. CPS and β-LS site-directed mutagenesis (Y345/8A, Y345/8F, E380/2D, E380/2Q, E380A) resulted in the reduction of their catalytic efficiency, with Y345A, E380A, and E382Q producing undetectable amounts of β-lactam product. However, [ 32 P]PP i -ATP exchange assays demonstrated Y345A and E380A undergo the first half-reaction, with the remaining active mutants showing decreased forward commitment to β-lactam cyclization. pH-rate profiles of CPS and β-LS supported the importance of a Tyr-Glu dyad in β-lactam formation and suggested its reverse protonation in β-LS. The kinetics of CPS double-site mutants reinforced the synergism of Tyr-Glu in catalysis. Furthermore, significant solvent isotope effects on k cat ( D k cat ) for Y345F (1.9) and Y348F (1.7) maintained the assignment of Y345/8 in proton transfer. A proton inventory on Y348F determined its D (k cat /K m ) = 0.2 to arise from multiple reactant-state fractionation factors, presumably from water molecule(s) replacing the missing Tyr hydroxyl. The role of a CPS and β-LS Tyr-Glu catalytic dyad was solidified by a significant decrease in mutant k cat viscosity dependence with respect to the wild-type enzymes. The evolutionary relation and potential for engineered biosynthesis were demonstrated by β-LS acting as a carbapenam synthetase.The compilation of genome sequence information has revealed genes encoding proteins of natural product biosynthetic pathways that differ in function from their primary metabolic ancestors. The clear evolution of these often well-studied enzymes to new, specialized roles is striking but frequently difficult to deduce from their primary sequence alone (1). Particularly in microorganisms, it is thought that survival under environmental stress will select mutant organisms whose overall fitness has improved (2,3). The β-lactam-containing secondary metabolites, most widely known for their antibacterial properties, are an instructive example of this evolutionary process.*To whom correspondence should be addressed. Phone: (410) . ctownsend@jhu.edu. ‖ Co-first authors equally contributed to the manuscript Supporting Information Available: Tables of all primers used to obtain the CPS and β-LS mutants, a table of CPS and β-LS viscosity dependence results, UV-vis data with CPS Y345A and E380A with and without hydroxylamine, proton inventory linear plot, [ 32 P] PP i -ATP exchange data, and nitrocefin assay result along with the corresponding expression SDS-PAGE gel from reactions with β-LS/ ATP and CMPr. This material is available free ...
β-Lactam synthetase (β-LS) is the paradigm of a growing class of enzymes that form the critical β-lactam ring in the clavam and carbapenem antibiotics. β-LS catalyzes a two stage reaction in which N2-(2-carboxyethyl)-l-arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to β-lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. β-Lactam formation was evaluated on the basis of X-ray crystal structures, site-specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate pre-organization, a well-stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four-membered ring formation to the magnitude of a protein conformational change.
The 2-azetidinone ring of the Class A and D β-lactamase inhibitor clavulanic acid (1) is synthesized by the ATP-utilizing enzyme β-lactam synthetase (βLS). A hydroxyethyl group attached to C-6 of 1 in the (S) configuration markedly enhances the efficacy of this compound against Class C β-lactamases. Guided by a series of X-ray structures of βLS, we have engineered this enzyme to act upon a methylated substrate analogue to give selectively the (3S)-methyl β-lactam core, which, upon closure of the second ring of the bicyclic system of 1, would lead to the (6S)-methylated clavulanic acid derivative.
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