Efficient Inhibition of Class A and Class D beta-Lactamases by Michaelis Complexes (original) (raw)
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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.
Journal of the American Chemical Society, 2012
The class D β-lactamases are characterized by the presence of a carboxylated lysine in the active site that participates in catalysis. Found in Acinetobacter baumannii, OXA-24 is a class D carbapenem hydrolyzing enzyme that exhibits resistance to most available β-lactamase inhibitors. In this study, the reaction between a 6-alkylidiene penam sulfone inhibitor, SA-1-204, in single crystals of OXA-24 is followed by Raman microscopy. Details of its reaction with SA-1-204 provide insight into the enzyme's mode of action and help define the mechanism of inhibition. When the crystal is maintained in HEPES buffer, the reaction is fast, shorter than the time scale of the Raman experiment. However, when the crystal holding solution contains 28% PEG 2000, the reaction is slower and can be recorded by Raman microscopy in real time; the inhibitor's Raman bands quickly disappear, transient features are seen due to an early intermediate, and, at approximately 2 to 11 minutes, new bands appear that are assigned to the late intermediate species. At about 50 minutes, bands due to all intermediates are replaced by Raman signals of the unreacted inhibitor. The new population remains unchanged indicating i) that the OXA-24 is no longer active and ii) that the decarboxylation of Lys84 occurred during the first reaction cycle. Using absorbance spectroscopy, a one cycle reaction could be carried out in aqueous solution producing inactive OXA-24 as assayed by the chromogenic substrate nitrocefin. However, activity could be restored by reacting aqueous OXA-24 with a large excess of NaHCO 3 which recarboxylates Lys84. In contrast the addition of NaHCO 3 was not successful in reactivating OXA-24 in the crystalline state; this is ascribed to the inability to create a concentration of NaHCO 3 in large excess over the OXA-24 that is present in the crystal. The finding that inhibitor
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.
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.
Biochemistry, 2015
For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH2OH group (6β-(hydroxymethyl)penicillanic acid). The 6β substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(hydroxymethyl) sulbactam. Rapid mix-rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6β-(hydroxymethyl) sulbactam formed long...
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.
Journal of Biological Chemistry, 2009
In an effort to devise strategies for overcoming bacterial -lactamases, we studied LN-1-255, a 6-alkylidene-2-substituted penicillin sulfone inhibitor. By possessing a catecholic functionality that resembles a natural bacterial siderophore, LN-1-255 is unique among -lactamase inhibitors. LN-1-255 combined with piperacillin was more potent against Escherichia coli DH10B strains bearing bla SHV extended-spectrum and inhibitor-resistant -lactamases than an equivalent amount of tazobactam and piperacillin. In addition, LN-1-255 significantly enhanced the activity of ceftazidime and cefpirome against extended-spectrum cephalosporin and Sme-1 containing carbapenem-resistant clinical strains. LN-1-255 inhibited SHV-1 and SHV-2 -lactamases with nM affinity (K I ؍ 110 ؎ 10 and 100 ؎ 10 nM, respectively). When LN-1-255 inactivated SHV -lactamases, a single intermediate was detected by mass spectrometry. The crystal structure of LN-1-255 in complex with SHV-1 was determined at 1.55 Å resolution. Interestingly, this novel inhibitor forms a bicyclic aromatic intermediate with its carbonyl oxygen pointing out of the oxyanion hole and forming hydrogen bonds with Lys-234 and Ser-130 in the active site. Electron density for the "tail" of LN-1-255 is less ordered and modeled in two conformations. Both conformations have the LN-1-255 carboxyl group interacting with Arg-244, yet the remaining tails of the two conformations diverge. The observed presence of the bicyclic aromatic intermediate with its carbonyl oxygen positioned outside of the oxyanion hole provides a rationale for the stability of this inhibitory intermediate. The 2-substituted penicillin sulfone, LN-1-255, is proving to be an important lead compound for novel -lactamase inhibitor design.
Computational and Structural Biotechnology Journal, 2021
AmpC BER is an extended-spectrum (ES) class C b-lactamase with a two-amino-acid insertion in the H10 helix region located at the boundary of the active site compared with its narrow spectrum progenitor. The crystal structure of the wild-type AmpC BER revealed that the insertion widens the active site by restructuring the flexible H10 helix region, which is the structural basis for its ES activity. Besides, two sulfates originated from the crystallization solution were observed in the active site. The presence of sulfatebinding subsites, together with the recognition of ring-structured chemical scaffolds by AmpC BER, led us to perform in silico molecular docking experiments with halisulfates, natural products isolated from marine sponge. Inspired by the snug fit of halisulfates within the active site, we demonstrated that halisulfate 3 and 5 significantly inhibit ES class C b-lactamases. Especially, halisulfate 5 is comparable to avibactam in terms of inhibition efficiency; it inhibits the nitrocefin-hydrolyzing activity of AmpC BER with a K i value of 5.87 lM in a competitive manner. Furthermore, halisulfate 5 displayed moderate and weak inhibition activities against class A and class B/D enzymes, respectively. The treatment of blactamase inhibitors (BLIs) in combination with b-lactam antibiotics is a working strategy to cope with infections by pathogens producing ES b-lactamases. Considering the emergence and dissemination of enzymes insensitive to clinically-used BLIs, the broad inhibition spectrum and structural difference of halisulfates would be used to develop novel BLIs that can escape the bacterial resistance mechanism mediated by b-lactamases.
Journal of the American Chemical Society, 2013
Bacteria that cause most of the hospital-acquired infections make use of class C β-lactamase (CBL) among other enzymes to resist a wide spectrum of modern antibiotics and pose a major public health concern. Other than the general features, details of the defensive mechanism by CBL, leading to the hydrolysis of drug molecules, remain a matter of debate, in particular, the identification of the general base, and role of active site residues and substrate. In an attempt to unravel the detailed molecular mechanism, we carried out extensive hybrid quantum mechanical/molecular mechanical (QM/MM) Car-Parrinello molecular dynamics (MD) simulation of the reaction with the aid of the metadynamics technique. Based on that we report here the mechanism of the formation of the acyl-enzyme complex from the Henry-Michaelis complex formed by β-lactam antibiotics and CBL. We considered two β-lactam antibiotics, namely cephalothin and aztreonam, belonging to two different subfamilies. A general mechanism for the formation of β-lactam antibiotic-CBL acyl-enzyme complex is elicited and the individual roles of the active site residues and substrate are probed. General base in the acylation step has been identified as Lys 67 while Tyr 150 aids the protonation of β-lactam nitrogen through, either the substrate carboxylate group, or a water molecule.
Penicillin Sulfone Inhibitors of Class D -Lactamases
Antimicrobial Agents and Chemotherapy, 2010
OXA -lactamases are largely responsible for -lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the -lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D -lactamases. To overcome this challenge, we explored the abilities of -lactamase inhibitors of the C-2-and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D -lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K i values for the OXA-1 -lactamase (0.70 ؎ 0.14 3 1.60 ؎ 0.30 M) and demonstrated significant K i improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K i s for the OXA-10, -14, -17, and -24/40 -lactamases (0.20 ؎ 0.04 3 17 ؎ 4 M). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.