Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

Vandavasi, Venu Gopal; Langan, Patricia S.; Weiss, Kevin L.; Parks, Jerry M.; Cooper, J.B.; Ginell, Stephan L.; Coates, Leighton

doi:10.1128/aac.01636-16

Cited by 32 publications

(29 citation statements)

References 40 publications

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“…As the pH 6.3 structure is identical to the pH 7.9 complex, this indicates the pK a of Lys73 is likely between pH 5.3 and 6.3, significantly lower than the estimate of 8.0 to 8.5 in previous studies of β-lactam hydrolysis (41). Additionally, computational predictions indicated similar disparity in Lys73 pK a between the acyl-enzyme complexes of avibactam and β-lactam inhibitor cefoxitin (6.24 versus 7.82; SI Appendix, Table S2) (11,42). Although these calculations were performed without using thermodynamic integration based on extensive explicit-solvent molecular dynamics simulations as done by others and were thus less accurate (41), they suggested that it would be of interest to further probe Lys73 pK a .…”

Section: Resultsmentioning

confidence: 66%

Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam

Pemberton

Noor

Kumar

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Gram-negative bacteria expressing class A β-lactamases pose a serious health threat due to their ability to inactivate all β-lactam antibiotics. The acyl–enzyme intermediate is a central milestone in the hydrolysis reaction catalyzed by these enzymes. However, the protonation states of the catalytic residues in this complex have never been fully analyzed experimentally due to inherent difficulties. To help unravel the ambiguity surrounding class A β-lactamase catalysis, we have used ultrahigh-resolution X-ray crystallography and the recently approved β-lactamase inhibitor avibactam to trap the acyl–enzyme complex of class A β-lactamase CTX-M-14 at varying pHs. A 0.83-Å-resolution CTX-M-14 complex structure at pH 7.9 revealed a neutral state for both Lys73 and Glu166. Furthermore, the avibactam hydroxylamine-O-sulfonate group conformation varied according to pH, and this conformational switch appeared to correspond to a change in the Lys73 protonation state at low pH. In conjunction with computational analyses, our structures suggest that Lys73 has a perturbed acid dissociation constant (pKa) compared with acyl–enzyme complexes with β-lactams, hindering its function to deprotonate Glu166 and the initiation of the deacylation reaction. Further NMR analysis demonstrated Lys73 pKato be ∼5.2 to 5.6. Together with previous ultrahigh-resolution crystal structures, these findings enable us to follow the proton transfer process of the entire acylation reaction and reveal the critical role of Lys73. They also shed light on the stability and reversibility of the avibactam carbamoyl acyl–enzyme complex, highlighting the effect of substrate functional groups in influencing the protonation states of catalytic residues and subsequently the progression of the reaction.

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Section: Resultsmentioning

confidence: 66%

Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam

Pemberton

Noor

Kumar

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Overall, our structures reveal that Ser70 is primed by deprotonation for nucleophilic attack on the AVI C-7 atom. It is still uncertain if this deprotonation is driven by Lys73 or Glu166 via a catalytic water that bridges both Glu166 and Ser70 (17)(18)(19)(20)(21)(22). Using both highresolution structural studies and molecular dynamics simulations, both Lys73 and Ser130 are also believed to participate in a proton shuttle resulting in the protonation of the AVI N-6 in the acylation step (15,23).…”

Section: Resultsmentioning

confidence: 99%

Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam

Ruggiero

Papp-Wallace

Brunetti

et al. 2019

Antimicrob Agents Chemother

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The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-β-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D β-lactamases (i.e., k2/K of ≈103 M−1 s−1) but lower than other class A β-lactamases (i.e., k2/K = 104 to 105 M−1 s−1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K.) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum β-lactamases (ESBLs). Comparatively, relebactam combined with a β-lactam is more potent against Escherichia coli producing PER-2 variants than β-lactam–AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

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“…Additionally, the study by Nichols et al (28) observed a proton transfer from Ser-70 to Glu-166 in the active site of class A ␤-lactamase CTX-M upon binding of a non-electrophilic ligand, lending experimental support to this model. Very recently, Vandavasi et al (29) reported for the first time structures of CTX-M in the acyl-enzyme complex with a monobactam, with Lys-73 indeed deprotonated to serve as general base for the acylation reaction. All these results support the model that the protonated Lys-73 in the apo-state can become deprotonated upon substrate binding to initiate the acylation reaction.…”

Section: Discussionmentioning

confidence: 99%

Crystallographic Snapshots of Class A β-Lactamase Catalysis Reveal Structural Changes That Facilitate β-Lactam Hydrolysis

Pan

Lei

et al. 2017

Journal of Biological Chemistry

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β-Lactamases confer resistance to β-lactam-based antibiotics. There is great interest in understanding their mechanisms to enable the development of β-lactamase-specific inhibitors. The mechanism of class A β-lactamases has been studied extensively, revealing Lys-73 and Glu-166 as general bases that assist the catalytic residue Ser-70. However, the specific roles of these two residues within the catalytic cycle remain not fully understood. To help resolve this, we first identified an E166H mutant that is functional but is kinetically slow. We then carried out time-resolved crystallographic study of a full cycle of the catalytic reaction. We obtained structures that represent apo, S*-acylation, andS*-deacylation states and analyzed the conformational changes of His-166. The "in" conformation in the apo structure allows His-166 to form a hydrogen bond with Lys-73. The unexpected "flipped-out" conformation of His-166 in the S*-acylation structure was further examined by molecular dynamics simulations, which suggested deprotonated Lys-73 serving as the general base for acylation. The "revert-in" conformation in theS*-deacylation structure aligns His-166 toward the water molecule that hydrolyzes the acyl adduct. Finally, when the acyl adduct is fully hydrolyzed, His-166 rotates back to the "in" conformation of the apo-state, restoring the Lys-73/His-166 interaction. Using His-166 as surrogate, our study identifies distinct conformational changes within the active site during catalysis. We suggest that the native Glu-166 executes similar changes in a less constricted way. Taken together, this structural series improves our understanding of β-lactam hydrolysis in this important class of enzymes.

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Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

Cited by 32 publications

References 40 publications

Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam

Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam

Structural Insights into the Inhibition of the Extended-Spectrum β-Lactamase PER-2 by Avibactam

Crystallographic Snapshots of Class A β-Lactamase Catalysis Reveal Structural Changes That Facilitate β-Lactam Hydrolysis

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