Detecting a Quasi-stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone (original) (raw)
Related papers
Journal of the American Chemical Society, 2009
Tazobactam, sulbactam, and clavulanic acid are the only β-lactamase inhibitors in clinical use. Comparative inhibitory activities of clavulanic acid, sulbactam, and tazobactam against clinically important β-lactamases conclude that tazobactam is superior to both clavulanic acid and sulbactam. Thus far, the majority of explanations for this phenomenon have relied on kinetic studies, which report differences in the ligands' apparent dissociation constants and number of turnovers before inactivation. Due their innate limitations, these investigations do not examine the identity of intermediates on the reaction pathway and relate them to the efficacy of the inhibitors. In the present study, the reactions between the three inhibitors and SHV-1 β-lactamase have been examined in single crystals using a Raman microscope. The results show that tazobactam forms a predominant population of trans-enamine, a chemically-inert species, with SHV-1 while clavulanate and sulbactam form a mixture of trans-enamine and two labile species, the cis-enamine and imine. The same reactions are then reexamined using a deacylation-deficient variant, SHV E166A, that has been used to trap acyl-enzyme intermediates for X-ray crystallographic analysis. Our Raman data show that significant differences exist between the wild-type and SHV E166A acyl-enzyme populations. Namely, compared to SHV-1, sulbactam shows significantly smaller populations of cis-enamine and imine in the E166A variant while clavulanate exists almost exclusively as trans-enamine in the E166A active site. Using clavulanate as an example, we also show that Raman crystallography can provide novel information on the presence of multiple conformers or tautomers for intermediates within a complex reaction pathway. These insights caution against the interpretation of experimental data obtained with deacylation-deficient β-lactamases to make mechanistic conclusions about inhibitors within the enzyme.
Sulbactam Forms Only Minimal Amounts of Irreversible Acrylate-Enzyme with SHV-1 β-Lactamase †
Biochemistry, 2007
Sulbactam is a mechanism-based inhibitor of β-lactamase enzymes used in clinical practice. It undergoes a complex series of chemical reactions in the active site that have been studied extensively in the past three decades. However, the actual species that gives rise to inhibition in a clinical setting has not been established. Recent studies by our group, using Raman microscopy and X-ray crystallography, have found that large quantities of enamine-based acyl-enzyme species are present within minutes in single crystals of SHV-1 β-lactamases which can lead to significant inhibition. The enamines are formed by break down of the cyclic beta-lactam structures with further transformations leading to imine formation and subsequent isomerization to cis and/or trans enamines. Another favored form of inhibition arises from attack on the imine by a second nucleophilic amino acid side chain, e.g. from serine 130, to form a cross linked species in the active site that can degrade to an acrylate-like species irreversibly bound to the enzyme. Thus, the imine is at a branch point on the reaction pathway. Using sulbactam and 6,6-dideuterated sulbactam we follow these alternate paths in WT and E166A SHV-1 β-lactamase by means of Raman microscopic studies on single enzyme crystals. For the unlabeled sulbactam, the Raman data show the presence of an acrylate-like species, probably 3-serine acrylate, several hours after the reaction is started in the crystal. However, for the 6,6 dideutero analog the acrylate signature appears on the time scale of minutes. The Raman signatures, principally an intense feature near 1530 cm −1 , are assigned based on quantum mechanical calculations on model compounds that mimic acrylate species in the active site. The different time scales observed for acrylate-like product formation are ascribed to different rates of reaction involving the imine intermediate. It is proposed that for the unsubstituted sulbactam the conversion from imine to enamine, which involves breaking a C-H bond, is aided by quantum mechanical tunneling. For the 6,6 dideutero-sulbactam the same step involves breaking a C-D bond, which has little or no assistance from tunneling. Consequently the conversion to enamines is slower, a higher population of imine results, presenting the opportunity for the competing reaction with the second nucleophile, serine 130 being the prime candidate. The hydrolysis of the resulting cross-linked intermediate leads to the observed rapid build-up of the acrylate product in the Raman spectra from the dideutero-analog. The protocol used here, essentially running the reactions with the two forms of sulbactam in parallel, provides an element of control and enables us to conclude that, for the unsubstituted sulbactam, the formation of the cross linked intermediate and the final irreversible acrylate product is not a significant route to inhibition of SHV-1.
Biochemistry, 2008
The clinically used inhibitors tazobactam and sulbactam are effective in the inhibition of activity of class A -lactamases, but not for class D -lactamases. The two inhibitors exhibit a complex multistep profile for their chemistry of inhibition with class A -lactamases. To compare the inhibition profiles for class A and D enzymes, the reactions were investigated within OXA-10 -lactamase (a class D enzyme) crystals using a Raman microscope. The favored reaction pathway appears to be distinctly different from that for class A -lactamases. In contrast to the case of class A enzymes that favor the formation of a key enamine species, the OXA-10 enzyme forms an R, -unsaturated acrylate (acid or ester). Quantum mechanical calculations support the likely product as the adduct of Ser115 to the acrylate. Few enaminelike species are formed by sulbactam or tazobactam with this enzyme. Taken together, our results show that the facile conversion of the initial imine, formed upon acylation of the active site Ser67, to the cisand/or trans-enamine is disfavored. Instead, there is a significant population of the imine that could either experience cross-linking to a second nucleophile (e.g., Ser115) or give rise to the R, -unsaturated product and permanent inhibition. Alternatively, the imine can undergo hydrolysis to regenerate the catalytically active OXA-10 enzyme. This last process is the dominant one for class D -lactamases since the enzyme is not effectively inhibited. In contrast to sulbactam and tazobactam, the reactions between oxacillin or 6R-hydroxyisopropylpenicillinate (both substrates) and OXA-10 -lactamase appear much less complex. These compounds lead to a single acyl-enzyme species, the presence of which was confirmed by Raman and MALDI-TOF experiments.
PLoS ONE, 2014
b-Lactamases are the major reason b-lactam resistance is seen in Gram-negative bacteria. To combat this resistance mechanism, b-lactamase inhibitors are currently being developed. Presently, there are only three that are in clinical use (clavulanate, sulbactam and tazobactam). In order to address this important medical need, we explored a new inhibition strategy that takes advantage of a long-lived inhibitory trans-enamine intermediate. SA2-13 was previously synthesized and shown to have a lower k react than tazobactam. We investigated here the importance of the carboxyl linker length and composition by synthesizing three analogs of SA2-13 (PSR-4-157, PSR-4-155, and PSR-3-226). All SA2-13 analogs yielded higher turnover numbers and k react compared to SA2-13. We next demonstrated using protein crystallography that increasing the linker length by one carbon allowed for better capture of a trans-enamine intermediate; in contrast, this trans-enamine intermediate did not occur when the C2 linker length was decreased by one carbon. If the linker was altered by both shortening it and changing the carboxyl moiety into a neutral amide moiety, the stable trans-enamine intermediate in wt SHV-1 did not form; this intermediate could only be observed when a deacylation deficient E166A variant was studied. We subsequently studied SA2-13 against a relatively recently discovered inhibitor-resistant (IR) variant of SHV-1, SHV K234R. Despite the alteration in the mechanism of resistance due to the KRR change in this variant, SA2-13 was effective at inhibiting this IR enzyme and formed a trans-enamine inhibitory intermediate similar to the intermediate seen in the wt SHV-1 structure. Taken together, our data reveals that the C2 side chain linker length and composition profoundly affect the formation of the trans-enamine intermediate of penam sulfones. We also show that the design of SA2-13 derivatives offers promise against IR SHV b-lactamases that possess the K234R substitution.
mBio, 2022
β-Lactamases hydrolyze β-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) β-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum β-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a D-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cb closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of β-lactamase inhibition by these agents. β-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing β-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to β-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining β-lactams with β-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to β-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of β-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.
Antimicrobial Agents and Chemotherapy
Sulbactam is one of four β-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of β-lactam antibiotics by these bacterial enzymes. As a β-lactam itself, sulbactam is susceptible to degradation by β-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 β-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the β-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A β-lactamases varied widely in their susceptibility to inhibition, the cla...
Raman Spectra of Interchanging β-Lactamase Inhibitor Intermediates on the Millisecond Time Scale
Journal of the American Chemical Society, 2013
Rapid mix-rapid freeze is a powerful method to study the mechanisms of enzyme-substrate reactions in solution. Here we report a protocol that combines this method with normal (nonresonance) Raman microscopy to enable us to define molecular details of intermediates at early time points. With this combined method, SHV-1, a class A β-lactamase, and tazobactam, a commercially available β-lactamase inhibitor, were rapidly mixed on the millisecond timescale , then were flash-frozen by injecting into an isopentane solution surrounded by liquid nitrogen. The "ice" was finally freeze-dried and characterized by Raman microscopy. We found that, in solution at 25 milliseconds, the reaction is almost complete giving rise to a major population composed of the trans-enamine intermediate. Between 25-500 milliseconds, minor populations of protonated imine are detected, that have previously been postulated to precede enamine intermediates. However, within 1 second, the imines are converted entirely to enamines. Interestingly, with this method, we can measure directly the turnover number of SHV-1 and tazobactam. At 1 : 4 ratio (enzyme : inhibitor) or greater, the enzyme is completely inhibited, a number that agrees with the turnover number derived from steady-state kinetic methods. This application, employing nonintensity enhanced Raman spectroscopy, provides a general and effective route to study the early events in enzyme-substrate reactions.
Efficient Inhibition of Class A and Class D beta-Lactamases by Michaelis Complexes
Journal of Biological Chemistry, 2007
A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 -lactamases with K I ؍ 42 ؎ 4 nM and 1.0 ؎ 0.1 M, respectively. To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution. Raman signatures characteristic of the unreacted -lactam ring show that in both phases the inhibitor binds as a noncovalent Michaelis-like complex. This complex is present as the major population for periods of up to an hour. On longer time scales, the Raman data show that -lactam ring opening eventually leads to a complex mixture of reaction products. However, the data clearly demonstrate that the key species for inhibition on the time scale of bacterial half-lives is the noncovalent complex preceding acylation.
PLoS ONE, 2012
Bacterial b-lactamase enzymes are in large part responsible for the decreased ability of b-lactam antibiotics to combat infections. The inability to overcome b-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, ''suicide-type'' inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of b-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine b-lactamases.