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• W3087950656 abstract "An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase–catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism. An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase–catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism. The most important identified mechanism of resistance to the clinically important β-lactam–containing antibiotics likely involves their degradation by β-lactamases (1Bush K. Bradford P.A. Interplay between β-lactamases and new β-lactamase inhibitors.Nat. Rev. Microbiol. 2019; 17 (30837684): 295-30610.1038/s41579-019-0159-8Crossref PubMed Scopus (136) Google Scholar, 2Bush K. Past and present perspectives on β-lactamases.Antimicrob. Agents Chemother. 2018; 62 (30061284): e01076-1810.1128/AAC.01076-18Crossref PubMed Scopus (208) Google Scholar). There are two mechanistic classes of β-lactamases, i.e. the serine β-lactamases (SBLs) and the zinc ion–dependent metallo-β-lactamases. The SBLs are presently more important from a clinical perspective and are subdivided into Ambler classes A, C, and D, with the metallo-β-lactamases forming class B (3Hall B.G. Barlow M. Revised Ambler classification of β-lactamases.J. Antimicrob. Chemother. 2005; 55 (15872044): 1050-105110.1093/jac/dki130Crossref PubMed Scopus (113) Google Scholar, 4Philippon A. Jacquier H. Ruppé E. Labia R. Structure-based classification of class A β-lactamases, an update.Curr. Res. Transl. Med. 2019; 67 (31155436): 115-12210.1016/j.retram.2019.05.003Crossref PubMed Scopus (5) Google Scholar). Of the SBLs, the highly diverse class D SBLs are of increasing concern. Some class D SBLs efficiently catalyze the degradation of carbapenems, a class of β-lactams often used as last resort drugs because of their breadth of antibacterial activity and resistance to degradation by some SBLs (5Pitout J.D.D. Peirano G. Kock M.M. Strydom K.A. Matsumura Y. The global ascendency of OXA-48–type carbapenemases.Clin. Microbiol. Rev. 2019; 33 (31722889): e00102-1910.1128/CMR.00102-19Crossref PubMed Scopus (84) Google Scholar, 6Poirel L. Potron A. Nordmann P. OXA-48-like carbapenemases: the phantom menace.J. Antimicrob. Chemother. 2012; 67 (22499996): 1597-160610.1093/jac/dks121Crossref PubMed Scopus (550) Google Scholar, 7Kopotsa K. Osei Sekyere J. Mbelle N.M. Plasmid evolution in carbapenemase-producing Enterobacteriaceae: a review.Ann. N.Y. Acad. Sci. 2019; 1457 (31469443): 61-9110.1111/nyas.14223Crossref PubMed Scopus (31) Google Scholar, 8Evans B.A. Amyes S.G.B. OXA β-lactamases.Clin. Microbiol. Rev. 2014; 27 (24696435): 241-26310.1128/CMR.00117-13Crossref PubMed Scopus (394) Google Scholar). SBL catalysis proceeds via a hydrolytically labile acyl-enzyme complex (AEC) intermediate, formed by addition–elimination reaction of the nucleophilic serine residue (Ser70 in class D SBL OXA-48) with a substrate β-lactam (Fig. 1) (1Bush K. Bradford P.A. Interplay between β-lactamases and new β-lactamase inhibitors.Nat. Rev. Microbiol. 2019; 17 (30837684): 295-30610.1038/s41579-019-0159-8Crossref PubMed Scopus (136) Google Scholar, 9Matagne A. Dubus A. Galleni M. Frère J.-M. The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity.Nat. Prod. Rep. 1999; 16 (10101880): 1-1910.1039/a705983cCrossref PubMed Scopus (180) Google Scholar). An analogous AEC is formed in the case of β-lactam antibiotic-mediated inhibition of their bacterial targets, penicillin-binding proteins. By contrast with penicillin-binding proteins, in the case of SBL catalysis, the AEC undergoes efficient hydrolysis (Fig. 1A). Uniquely among studied SBL classes, the class D SBLs employ a carbamylated lysine for general acid/base catalysis (Fig. 1B) (10Llarrull L.I. Testero S.A. Fisher J.F. Mobashery S. The future of the β-lactams.Curr. Opin. Microbiol. 2010; 13 (20888287): 551-55710.1016/j.mib.2010.09.008Crossref PubMed Scopus (122) Google Scholar, 11Lohans C.T. Wang D.Y. Jorgensen C. Cahill S.T. Clifton I.J. McDonough M.A. Oswin H.P. Spencer J. Domene C. Claridge T.D.W. Brem J. Schofield C.J. 13C-Carbamylation as a mechanistic probe for the inhibition of class D β-lactamases by avibactam and halide ions.Org. Biomol. Chem. 2017; 15 (28678295): 6024-603210.1039/c7ob01514cCrossref PubMed Google Scholar). All current carbapenem drugs have a C-6 hydroxyethyl side chain. Some carbapenems, e.g. imipenem, have a hydrogen substituent in the 1β position, whereas others, e.g. meropenem, have a 1β-methyl substituent, which hinders degradation by human renal dehydropeptidase-1 (Fig. 1) (12Zhanel G.G. Wiebe R. Dilay L. Thomson K. Rubinstein E. Hoban D.J. Noreddin A.M. Karlowsky J.A. Comparative review of the carbapenems.Drugs. 2007; 67 (17488146): 1027-105210.2165/00003495-200767070-00006Crossref PubMed Scopus (408) Google Scholar). The carbapenem C-6 hydroxyethyl side chain is proposed to help stabilize the SBL AEC by slowing its hydrolysis, thereby enabling partial carbapenem resistance to SBL degradation (9Matagne A. Dubus A. Galleni M. Frère J.-M. The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity.Nat. Prod. Rep. 1999; 16 (10101880): 1-1910.1039/a705983cCrossref PubMed Scopus (180) Google Scholar). In support of this proposal, crystallographic studies of the AEC formed between the class A SBL TEM-1 and the carbapenem, imipenem, suggest that the C-6 hydroxyethyl side chain interacts with the “hydrolytic” water molecule in the active site (13Maveyraud L. Mourey L. Kotra L.P. Pedelacq J.-D. Guillet V. Mobashery S. Samama J.-P. Structural basis for clinical longevity of carbapenem antibiotics in the face of challenge by the common class A β-lactamases from the antibiotic-resistant bacteria.J. Am. Chem. Soc. 1998; 120: 9748-975210.1021/ja9818001Crossref Scopus (119) Google Scholar); it is proposed that this hydrogen-bonding interaction disfavors nucleophilic attack of the water molecule. The classical mechanism of SBL catalysis has recently been extended in work showing that class D SBLs can isomerize 1β-methyl–substituted carbapenems to form β-lactones, in addition to hydrolysis products (Fig. 1), raising the possibility of nonhydrolytic SBL-mediated carbapenem resistance (14Lohans C.T. van Groesen E. Kumar K. Tooke C.L. Spencer J. Paton R.S. Brem J. Schofield C.J. A new mechanism for β-lactamases: class D enzymes degrade 1β-methyl carbapenems through lactone formation.Angew. Chem. Int. Ed. Engl. 2018; 57 (29236332): 1282-128510.1002/anie.201711308Crossref PubMed Scopus (15) Google Scholar, 15Lohans C.T. Freeman E.I. Groesen E. van, Tooke C.L. Hinchliffe P. Spencer J. Brem J. Schofield C.J. Mechanistic insights into β-lactamase–catalysed carbapenem degradation through product characterisation.Sci. Rep. 2019; 9 (31541180)13608 10.1038/s41598-019-49264-0Crossref PubMed Scopus (12) Google Scholar). The β-lactone products are proposed to be formed by initial nucleophilic attack of the C-6 hydroxyethyl hydroxyl group onto the ester carbonyl of the AEC. In support of this mechanism and arguing against their formation via reaction of a noncovalently bound complex, at least in some cases, the β-lactones can react reversibly with class D SBLs to give a covalently modified complex (14Lohans C.T. van Groesen E. Kumar K. Tooke C.L. Spencer J. Paton R.S. Brem J. Schofield C.J. A new mechanism for β-lactamases: class D enzymes degrade 1β-methyl carbapenems through lactone formation.Angew. Chem. Int. Ed. Engl. 2018; 57 (29236332): 1282-128510.1002/anie.201711308Crossref PubMed Scopus (15) Google Scholar). The carbapenem-derived β-lactones are less potent SBL inhibitors than the parent carbapenems, but with optimization to enable them to form stable acyl-enzyme complexes, β-lactones may form the basis of a new type of SBL inhibitor (14Lohans C.T. van Groesen E. Kumar K. Tooke C.L. Spencer J. Paton R.S. Brem J. Schofield C.J. A new mechanism for β-lactamases: class D enzymes degrade 1β-methyl carbapenems through lactone formation.Angew. Chem. Int. Ed. Engl. 2018; 57 (29236332): 1282-128510.1002/anie.201711308Crossref PubMed Scopus (15) Google Scholar). The factors determining bifurcation between hydrolysis and β-lactone formation are of interest from the perspectives of carbapenem antibiotic resistance and antibacterial drug development. Kinetic analyses show that the class D SBLs degrade 1β-methyl–substituted carbapenems more slowly than those with a 1β-hydrogen substituent, leading to the proposal that β-lactone formation may occur when hydrolysis is disfavored at the AEC (16Oueslati S. Nordmann P. Poirel L. Heterogeneous hydrolytic features for OXA-48–like β-lactamases.J. Antimicrob. Chemother. 2015; 70 (25583748): 1059-106310.1093/jac/dku524PubMed Google Scholar, 17Queenan A.M. Shang W. Flamm R. Bush K. Hydrolysis and inhibition profiles of β-lactamases from molecular classes A to D with doripenem, imipenem, and meropenem.Antimicrob. Agents Chemother. 2010; 54 (19884379): 565-56910.1128/AAC.01004-09Crossref PubMed Scopus (68) Google Scholar). Computational studies indicate that the carbapenem 1β-methyl substituent may influence the C-6 hydroxyethyl side chain conformation in a manner favoring β-lactone formation (or, at least, disfavoring hydrolysis) (14Lohans C.T. van Groesen E. Kumar K. Tooke C.L. Spencer J. Paton R.S. Brem J. Schofield C.J. A new mechanism for β-lactamases: class D enzymes degrade 1β-methyl carbapenems through lactone formation.Angew. Chem. Int. Ed. Engl. 2018; 57 (29236332): 1282-128510.1002/anie.201711308Crossref PubMed Scopus (15) Google Scholar). Crystallographic studies have shown that, in the AEC derived from class D SBLs, the carbapenem C-6 hydroxyethyl side chain is positioned in a hydrophobic pocket (18Akhter S. Lund B.A. Ismael A. Langer M. Isaksson J. Christopeit T. Leiros H.K.S. Bayer A. A focused fragment library targeting the antibiotic resistance enzyme oxacillinase-48: synthesis, structural evaluation and inhibitor design.Eur. J. Med. Chem. 2018; 145 (29348071): 634-64810.1016/j.ejmech.2017.12.085Crossref PubMed Scopus (16) Google Scholar, 19Schneider K.D. Karpen M.E. Bonomo R.A. Leonard D.A. Powers R.A. The 1.4 Å crystal structure of the class D β-lactamase OXA-1 complexed with doripenem.Biochemistry. 2009; 48 (19919101): 11840-1184710.1021/bi901690rCrossref PubMed Scopus (52) Google Scholar, 20Stewart N.K. Smith C.A. Antunes N.T. Toth M. Vakulenko S.B. Role of the hydrophobic bridge in the carbapenemase activity of class D β-lactamases.Antimicrob. Agents Chemother. 2018; 63: e02191-1810.1128/AAC.02191-18Crossref Scopus (11) Google Scholar). In OXA-48, one of the most important class D SBLs from a clinical perspective (5Pitout J.D.D. Peirano G. Kock M.M. Strydom K.A. Matsumura Y. The global ascendency of OXA-48–type carbapenemases.Clin. Microbiol. Rev. 2019; 33 (31722889): e00102-1910.1128/CMR.00102-19Crossref PubMed Scopus (84) Google Scholar), this pocket is formed by the side chains of the highly, but not universally, conserved residues Val120, Trp105, and Leu158 (Fig. 1B) (18Akhter S. Lund B.A. Ismael A. Langer M. Isaksson J. Christopeit T. Leiros H.K.S. Bayer A. A focused fragment library targeting the antibiotic resistance enzyme oxacillinase-48: synthesis, structural evaluation and inhibitor design.Eur. J. Med. Chem. 2018; 145 (29348071): 634-64810.1016/j.ejmech.2017.12.085Crossref PubMed Scopus (16) Google Scholar). We report studies aimed at investigating the contribution of class D SBL residues involved in binding the carbapenem C-6 hydroxyethyl side chain on the kinetics and the reaction outcome of carbapenem degradation, focusing on the ratio of β-lactone to hydrolysis products formed. The results reveal that the identity of hydrophobic active site residues can affect the balance between carbapenem hydrolysis and isomerization to form β-lactone products. The clinically observed OXA-48 V120L variant (OXA-519) (21Dabos L. Bogaerts P. Bonnin R.A. Zavala A. Sacré P. Iorga B.I. Huang D.T. Glupczynski Y. Naas T. Genetic and biochemical characterization of OXA-519, a novel OXA-48–like β-lactamase.Antimicrob. Agents Chemother. 2018; 62: e00469-1810.1128/AAC.00469-18PubMed Google Scholar), which catalyzes carbapenem degradation more rapidly than the WT OXA-48, substantially favors β-lactone formation over hydrolysis. A similar increase in β-lactone production is found with an analogous OXA-23 variant (OXA-23 V128L) (8Evans B.A. Amyes S.G.B. OXA β-lactamases.Clin. Microbiol. Rev. 2014; 27 (24696435): 241-26310.1128/CMR.00117-13Crossref PubMed Scopus (394) Google Scholar). Importantly, in addition to the reported formation of β-lactone products from carbapenems with 1β-methyl substituents, we observed OXA-519 catalyzed formation of β-lactones from imipenem and panipenem, which have a 1β-hydrogen substituent. The roles of the OXA-48 residues surrounding the carbapenem C-6 hydroxyethyl side chain (i.e. Val120, Leu158, and Trp105) were investigated by mostly conservative substitutions, aiming to alter the carbapenem C-6 hydroxyethyl side chain conformation while maintaining catalytic activity and the overall protein fold. Thus, recombinant forms of the OXA-48 V120L, V120I, L158V, L158I, W105A, and W105F variants were produced and highly purified (Fig. S1). Comparison of recombinant WT OXA-48 with these six OXA-48 variants by CD spectroscopy and thermal melting analyses indicates that the substitutions do not have a substantial impact on the overall fold (Fig. S1). To investigate the impact of the six OXA-48 substitutions on catalytic activity, we initially carried out kinetic analyses monitoring product formation with the cephalosporin nitrocefin and substrate depletion with the two carbapenems meropenem and imipenem (Table 1). The carbapenems employed comprised one with (meropenem) and one without (imipenem) a 1β-methyl substituent. In all cases, the OXA-48 variants were reduced in their catalytic activity for nitrocefin and both carbapenems compared with WT OXA-48, as judged by kcat/Km values.Table 1Steady-state kinetic analyses of wildtype OXA-48 and variants with nitrocefin, meropenem, and imipenem, as determined spectrophotometrically. Assays were performed using nitrocefin (5–1500 μm) and enzyme (25–500 pm) in 50 mm sodium phosphate, pH 7.5, or meropenem (1.75–500 μm), or imipenem (5–500 μm) with enzyme (1.5–400 nm) in 50 mm sodium phosphate, pH 7.5, supplemented with 50 mm sodium bicarbonate. Kinetic parameters are means ± standard deviations (n = 3)OXA-48SubstratekcatKmkcat/Kms−1μm(m−1 s−1) × 10−6WTNitrocefin593.2 ± 18.323.9 ± 3.524.8 ± 3.7Meropenem0.087 ± 0.01<1.9aThe Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.>0.045aThe Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.Imipenem11.4 ± 0.557.7 ± 9.30.20 ± 0.03V120LNitrocefin287.1 ± 11.7184.1 ± 22.81.5 ± 0.20Meropenem1.1 ± 0.0851.0 ± 11.80.021 ± 0.005Imipenem0.49 ± 0.0066.8 ± 0.60.071 ± 0.006V120INitrocefin323.2 ± 11.140.1 ± 4.98.1 ± 1.02Meropenem0.94 ± 0.0112.6 ± 1.40.075 ± 0.008Imipenem17.9 ± 1.4208.2 ± 45.20.086 ± 0.00W105FNitrocefin178.1 ± 16.7298.4 ± 37.80.60 ± 0.09Meropenem0.038 ± 0.0007<1.75aThe Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.>0.022aThe Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.W105ANitrocefin70.72 ± 1.5218.1 ± 9.50.32 ± 0.02MeropenembNot determined because of slow reaction rate.L158VNitrocefin54.9 ± 4.316.1 ± 3.33.4 ± 0.8Meropenem0.0081 ± 0.0002<2aThe Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.>0.0041aThe Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.L158INitrocefin59.8 ± 3.58.1 ± 2.27.4 ± 2.1Meropenem0.031 ± 0.0057.1 ± 4.10.0044 ± 0.003a The Km values shown are the lowest [S] at which turnover could be accurately measured. The actual Km values are thus lower and the kcat/Km values are larger than those given.b Not determined because of slow reaction rate. Open table in a new tab For all the OXA-48 variants, the results imply nitrocefin is a better substrate than the two carbapenems, as judged by kcat and kcat/Km values, although in some cases the Km values for the carbapenems are lower than for nitrocefin (Table 1). With nitrocefin, the Km values are increased for OXA-48 V120L, V120I, W105F, and W105A but are decreased for the L158V and L158I variants. Meropenem degradation by OXA-48 W105A was too slow for kinetic constants to be accurately determined. Mass spectrometric experiments suggest that the acylation of OXA-48 W105A by meropenem occurs efficiently (Fig. S2), implying that this substitution may disfavor deacylation of the AEC intermediate. Comparison of the results for the two carbapenems with the OXA-48 V120L and V120I variants reveals interesting variations in the kcat and Km values relative to WT OXA-48. The kcat values increase for meropenem with both the V120L and V120I variants and increases for imipenem with V120I variant but decreases for imipenem with the V120L variant. Compared with WT OXA-48, both the V120L and V120I variants catalyze meropenem degradation less efficiently than imipenem (as judged by kcat/Km values). These results reveal that interactions between specific active site residues and specific carbapenems can impact on kinetics; we hence envisaged that such interactions might alter the ratio of hydrolysis to β-lactone products. To investigate whether lysine carbamylation is substantially altered in the variants compared with WT OXA-48, WT OXA-48 and the V120L and V120I variants were treated with NaH13CO3, and the extent of carbamylation was monitored by 13C NMR (Fig. S3) (11Lohans C.T. Wang D.Y. Jorgensen C. Cahill S.T. Clifton I.J. McDonough M.A. Oswin H.P. Spencer J. Domene C. Claridge T.D.W. Brem J. Schofield C.J. 13C-Carbamylation as a mechanistic probe for the inhibition of class D β-lactamases by avibactam and halide ions.Org. Biomol. Chem. 2017; 15 (28678295): 6024-603210.1039/c7ob01514cCrossref PubMed Google Scholar). These results suggest that the carbamylation state of all three enzymes is approximately comparable. With added sodium bicarbonate, the kcat and Km values with nitrocefin for WT OXA-48 and the V120L and V120I variants all increased (Table S1). To explore the generality of these observations, the impact of substituting Val128 (equivalent to Val120 in OXA-48) on the activity of OXA-23, a clinically important class D SBL that shares 39% sequence identity with OXA-48, was also examined. The V128L and V128I variants of OXA-23 were produced and highly purified (Fig. S4) (22Cahill S.T. Cain R. Wang D.Y. Lohans C.T. Wareham D.W. Oswin H.P. Mohammed J. Spencer J. Fishwick C.W.G. McDonough M.A. Schofield C.J. Brem J. Cyclic boronates inhibit all classes of β-lactamases.Antimicrob. Agents Chemother. 2017; 61: e02260-1610.1128/AAC.02260-16Crossref PubMed Scopus (69) Google Scholar, 23van Groesen E. Lohans C.T. Brem J. Aertker K.M.J. Claridge T.D.W. Schofield C.J. 19F NMR monitoring of reversible protein post-translational modifications: class D β-lactamase carbamylation and inhibition.Chem. Eur. J. 2019; 25 (31310409): 11837-1184110.1002/chem.201902529Crossref PubMed Scopus (6) Google Scholar). With nitrocefin, the kcat/Km values for OXA-23 V128I and V128L are lower than for WT OXA-23, because the Km values are substantially higher for both variants. Imipenem is a better substrate than meropenem for WT OXA-23 and, to a lesser extent, for OXA-23 V128I based on kcat/Km values. However, imipenem and meropenem are similarly good substrates for OXA-23 V128L (Table S2). The overall kinetic analyses with the class D SBL variants reveal variations in both kcat and Km values for the three tested substrates. To investigate whether these variations correlate with the ratio of hydrolysis:β-lactone products formed, we carried out studies using 1H NMR (600 MHz) spectroscopy. This enables differentiation between the two product types, which is not possible when using standard UV-visible assays for carbapenem turnover (14Lohans C.T. van Groesen E. Kumar K. Tooke C.L. Spencer J. Paton R.S. Brem J. Schofield C.J. A new mechanism for β-lactamases: class D enzymes degrade 1β-methyl carbapenems through lactone formation.Angew. Chem. Int. Ed. Engl. 2018; 57 (29236332): 1282-128510.1002/anie.201711308Crossref PubMed Scopus (15) Google Scholar). We monitored product formation using the signal corresponding to the C-9 methyl group, which manifests distinctive resonances for the carbapenem starting material, the hydrolysis products, and the β-lactone products (1.22, 1.18, and 1.55 ppm, respectively) (14Lohans C.T. van Groesen E. Kumar K. Tooke C.L. Spencer J. Paton R.S. Brem J. Schofield C.J. A new mechanism for β-lactamases: class D enzymes degrade 1β-methyl carbapenems through lactone formation.Angew. Chem. Int. Ed. Engl. 2018; 57 (29236332): 1282-128510.1002/anie.201711308Crossref PubMed Scopus (15) Google Scholar, 15Lohans C.T. Freeman E.I. Groesen E. van, Tooke C.L. Hinchliffe P. Spencer J. Brem J. Schofield C.J. Mechanistic insights into β-lactamase–catalysed carbapenem degradation through product characterisation.Sci. Rep. 2019; 9 (31541180)13608 10.1038/s41598-019-49264-0Crossref PubMed Scopus (12) Google Scholar). It should be noted that, in principle, the hydrolysis and β-lactone products can be produced in two epimeric 1-pyrroline forms (i.e. (S)-Δ1 imine and (R)-Δ1 imine), as well as the tautomeric 2-pyrroline (Δ2 enamine) form (Fig. S5A). Recent work has indicated that the Δ2 enamine is likely the predominant nascent enzymatic product with most class D SBLs, including OXA-48 and OXA-23 (15Lohans C.T. Freeman E.I. Groesen E. van, Tooke C.L. Hinchliffe P. Spencer J. Brem J. Schofield C.J. Mechanistic insights into β-lactamase–catalysed carbapenem degradation through product characterisation.Sci. Rep. 2019; 9 (31541180)13608 10.1038/s41598-019-49264-0Crossref PubMed Scopus (12) Google Scholar). In the case of the products derived from 1β-methyl–substituted carbapenems, rapid tautomerization yields the (R)-Δ1 imine product as the kinetic imine product (which is likely, at least predominantly, a nonenzymatic product) with subsequent conversion to the more thermodynamically stable (S)-Δ1 imine product (Fig. S5B) (15Lohans C.T. Freeman E.I. Groesen E. van, Tooke C.L. Hinchliffe P. Spencer J. Brem J. Schofield C.J. Mechanistic insights into β-lactamase–catalysed carbapenem degradation through product characterisation.Sci. Rep. 2019; 9 (31541180)13608 10.1038/s41598-019-49264-0Crossref PubMed Scopus (12) Google Scholar). Under our standard NMR assay conditions with the 1β-methyl–substituted carbapenem meropenem, the (S)-Δ1 imine product was usually the major product form observed for both the hydrolysis and β-lactone products, within the time frames of our assays (Fig. S5). All the studied OXA-48 variants catalyze reaction of the 1β-methyl–substituted carbapenem, meropenem to form both β-lactone and hydrolysis products (Fig. 2A and Fig. S6). The OXA-48 V120I, W105F, L158V, and L158I variants produce more of the hydrolysis products than the β-lactones. Of all the tested enzymes, the V120I variant forms the lowest amount of β-lactone relative to hydrolysis products. Although the WT and W105A enzymes produce a small excess of β-lactone, the V120L variant, which is clinically observed (21Dabos L. Bogaerts P. Bonnin R.A. Zavala A. Sacré P. Iorga B.I. Huang D.T. Glupczynski Y. Naas T. Genetic and biochemical characterization of OXA-519, a novel OXA-48–like β-lactamase.Antimicrob. Agents Chemother. 2018; 62: e00469-1810.1128/AAC.00469-18PubMed Google Scholar), produces considerably more β-lactones than hydrolysis products. Testing of the OXA-48 V120L variant with other 1β-methyl–substituted carbapenems (ertapenem and biapenem) similarly shows that β-lactone formation is the predominant pathway under the tested conditions (Fig. S7). These observations reveal that substitutions other than those directly involved in the acid/base catalytic machinery can influence the ratio of hydrolysis to β-lactone formation. Previous experiments have indicated that WT OXA-48 does not form β-lactones from carbapenems with a 1β-hydrogen rather than a 1β-methyl substituent, such as in the clinically used drugs imipenem and panipenem (16Oueslati S. Nordmann P. Poirel L. Heterogeneous hydrolytic features for OXA-48–like β-lactamases.J. Antimicrob. Chemother. 2015; 70 (25583748): 1059-106310.1093/jac/dku524PubMed Google Scholar, 17Queenan A.M. Shang W. Flamm R. Bush K. Hydrolysis and inhibition profiles of β-lactamases from molecular classes A to D with doripenem, imipenem, and meropenem.Antimicrob. Agents Chemother. 2010; 54 (19884379): 565-56910.1128/AAC.01004-09Crossref PubMed Scopus (68) Google Scholar). However, upon analyzing the products of the reactions of OXA-48 V120L with imipenem and panipenem, we observed new products in the 1H NMR spectra that are not observed with WT OXA-48 (Fig. 2B and Fig. S6). Characterization of these products by NMR analyses reveals formation of the previously unobserved β-lactones derived from imipenem and panipenem, in addition to the established hydrolysis products (Tables S3 and S4 and Figs. S8–S15). This observation demonstrates that β-lactone formation by class D SBLs is not limited to 1β-methyl–substituted carbapenems. We then investigated whether the identity of the residue at position 120 (OXA-48 numbering) plays a role in determining the reaction outcome for other class D SBLs. With meropenem, the OXA-23 V128L variant was observed to favor β-lactone formation compared with WT OXA-23, similar to what was observed for WT OXA-48 and OXA-48 V120L (Fig. S16). As for the observations with OXA-48 V120L, OXA-23 V128L forms β-lactone products from imipenem and panipenem, both of which lack a 1β-methyl substituent (Fig. S17). However, the OXA-23 V128I variant does not favor hydrolysis over β-lactone formation in the manner that was observed for OXA-48 V120I (Fig. 2A and Fig. S16). Comparison of the ratios of hydrolysis:β-lactone products (Fig. 2A and Figs. S6, S7, S16, and S17) with the kinetic data for kcat, Km, and kcat/Km values (Table 1 and Table S2) for the different tested class D SBL variants does not reveal any clear relationships. This implies that the ratio of hydrolysis:β-lactone products is not simply related to binding efficiency or stability of the AEC but rather is a function of specific interactions between carbapenem-derived in" @default.
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