Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl–enzyme conformational dynamics to extend antibiotic resistance
2020; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1074/jbc.ra120.016461
ISSN1083-351X
AutoresCatherine L. Tooke, Philip Hinchliffe, Robert A. Bonomo, Christopher J. Schofield, Adrian J. Mulholland, James Spencer,
Tópico(s)Berberine and alkaloids research
ResumoClass A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl–enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than altered KM values. High-resolution crystal structures of ESOC acyl–enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl–enzyme intermediate. Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl–enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than altered KM values. High-resolution crystal structures of ESOC acyl–enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl–enzyme intermediate. Increasing antimicrobial resistance threatens global public health (1Rice L.B. Mechanisms of resistance and clinical relevance of resistance to beta-lactams, glycopeptides, and fluoroquinolones.Mayo Clinic Proc. 2012; 87: 198-208Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), exemplified by resistance to β-lactams (e.g. carbapenems and cephalosporins), the most prescribed antibiotics worldwide (2Tooke C. Hinchliffe P. Bragginton E. Colenso C.K. Hirvonen V. Takebayashi Y. Spencer J. β-Lactamases and β-lactamase inhibitors in the 21st Century.J. Mol. Biol. 2019; 431: 3472-3500Crossref PubMed Scopus (145) Google Scholar). 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The endemic status of KPC SBLs worldwide, together with their hydrolytic activity against a broad spectrum of β-lactams, makes this enzyme family a leading cause of carbapenem and other β-lactam failure in health care–associated infections. The KPC family now comprises up to 54 distinct enzymes (18Naas T. Oueslati S. Bonnin R.A. Dabos M.L. Zavala A. Dortet L. Retailleau P. Iorga B.I. Beta-lactamase database (BLDB) – structure and function.J. Enzyme Inhib. Med. Chem. 2017; 32: 917-919Crossref PubMed Scopus (138) Google Scholar) representing various insertions, deletions, and substitutions. The parent enzyme, KPC-2, efficiently hydrolyzes penicillins, cephalosporins, and carbapenems but poorly hydrolyzes the expanded-spectrum oxyimino-cephalosporin (ESOC), ceftazidime, particularly compared with the structurally similar cefotaxime (Fig. 1) (13Mehta S.C. Rice K. Palzkill T. 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Ceftazidime-avibactam (Avycaz): for the treatment of complicated intra-abdominal and urinary tract infections.P T. 2016; 41: 479-483PubMed Google Scholar, 27Shirley M. Ceftazidime-avibactam: a Review in the treatment of serious Gram-negative bacterial infections.Drugs. 2018; 78: 675-692Crossref PubMed Scopus (74) Google Scholar). KPC variants are the focus of increasing attention, particularly with respect to activity towards ceftazidime. The most comprehensive study to date investigated the activity and stability of the natural KPC variants 2 to 11 that possess single- or double-point mutations at four positions (M49I, P104R, P104L, V240G, V240A, and H274Y; Table S1 (13Mehta S.C. Rice K. Palzkill T. Natural variants of the KPC-2 carbapenemase have Evolved increased catalytic efficiency for ceftazidime hydrolysis at the cost of enzyme stability.PLoS Pathog. 2015; 11: e1004949Crossref PubMed Scopus (55) Google Scholar)). This identified differences in ceftazidime hydrolysis, with the variants KPC-4 (P104R and V240G substitutions) and KPC-10 (P104R and H274Y) exhibiting 50- and 75-fold increases in kcat/KM compared with KPC-2, resulting in 32- and 42-fold increases in the ceftazidime minimal inhibitory concentration when the respective variants are expressed in recombinant E. coli (13Mehta S.C. Rice K. Palzkill T. Natural variants of the KPC-2 carbapenemase have Evolved increased catalytic efficiency for ceftazidime hydrolysis at the cost of enzyme stability.PLoS Pathog. 2015; 11: e1004949Crossref PubMed Scopus (55) Google Scholar). A reduction in thermodynamic stability of these variants further indicated that increased ceftazidime hydrolysis may be at a stability cost (13Mehta S.C. Rice K. Palzkill T. 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Bhagwat S.S. et al.Strategic Approaches to Overcome resistance against Gram-negative pathogens using β-lactamase inhibitors and β-lactam Enhancers: activity of three novel Diazabicyclooctanes WCK 5153, Zidebactam (WCK 5107), and WCK 4234.J. Med. Chem. 2018; 61: 4067-4086Crossref PubMed Scopus (63) Google Scholar) and two with antibiotic hydrolysis products (33Pemberton O.A. Zhang X. Chen Y. Molecular basis of substrate recognition and product Release by the Klebsiella pneumoniae carbapenemase (KPC-2).J. Med. Chem. 2017; 60: 3525-3530Crossref PubMed Scopus (26) Google Scholar), their interactions with substrates are not well understood. In particular, the absence of structures for any acyl–enzyme complexes limits explanation of their unusually broad spectrum of activity, and of how naturally occurring point variants tune activity toward different substrates (13Mehta S.C. Rice K. Palzkill T. Natural variants of the KPC-2 carbapenemase have Evolved increased catalytic efficiency for ceftazidime hydrolysis at the cost of enzyme stability.PLoS Pathog. 2015; 11: e1004949Crossref PubMed Scopus (55) Google Scholar). Here we describe investigations of ESOC turnover by KPC-2 and its doubly substituted variant KPC-4 that seek to understand the basis for the enhanced ceftazidime turnover, and consequent ceftazidime resistance of producer bacteria, by the latter. Crystal structures of the ceftazidime and cefotaxime acyl-enzymes of KPC-2, and the ceftazidime acyl-enzyme of KPC-4, identify that KPC-2 undergoes more extensive rearrangement on ceftazidime acylation than does KPC-4; extended molecular dynamics (MD) simulations of the acyl–enzyme complexes identify a conformation of the Ω loop, favored in KPC-2, that orients E166, the general base for deacylation, away from the acyl–enzyme carbonyl, restricting deacylation. These results highlight the mobility of loops within and around the KPC active-site pocket and the importance of this to hydrolysis of the ESOC substrate ceftazidime. Effecting alterations to the dynamic properties of the acyl–enzyme intermediate then provides a mechanism by which naturally occurring enzyme variants can modulate specificity. To investigate activity differences between the naturally occurring KPC variants KPC-2 and KPC-4 (P104R:V240G), we first determined steady-state kinetic parameters for their hydrolysis of the oxyimino-cephalosporins ceftazidime and cefotaxime as well as the reporter substrate nitrocefin (34O'Callaghan C.H. Morris A. Kirby S.M. Shingler A.H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate.Antimicrob. Agents Chemother. 1972; 1: 283-288Crossref PubMed Scopus (1420) Google Scholar) (Table 1).Table 1Steady-state parameters for cephalosporin hydrolysis by KPC-2 and KPC-4SubstrateKinetic parameterKPC-2KPC-4Ceftazidimekcat (s−1)1.9 (0.12)81 (7.6)KM (μM)530 (70)640 (110)kcat/KM (μM−1 s−1)0.0035 (5.2 × 10−4)0.13 (0.025)Cefotaximekcat (s−1)76 (6.6)262 (32)KM (μM)200 (29)190 (49)kcat/KM (μM−1 s−1)0.38 (0.064)1.37 (0.039)Nitrocefinkcat (s−1)610 (9)170 (44)KM (μM)18 (5)59 (20)kcat/KM (μM−1 s−1)34 (9.3)3 (1.2)KPC, Klebsiella pneumoniae carbapenemase.Standard errors are provided in parentheses; n = 3; calculated in GraphPad Prism. Open table in a new tab KPC, Klebsiella pneumoniae carbapenemase. Standard errors are provided in parentheses; n = 3; calculated in GraphPad Prism. Consistent with previous reports of increased catalytic efficiency (kcat/KM) for ceftazidime hydrolysis by some KPC variants (13Mehta S.C. Rice K. Palzkill T. Natural variants of the KPC-2 carbapenemase have Evolved increased catalytic efficiency for ceftazidime hydrolysis at the cost of enzyme stability.PLoS Pathog. 2015; 11: e1004949Crossref PubMed Scopus (55) Google Scholar), we observe a 40-fold increase of kcat/KM for KPC-4 compared with KPC-2. Ceftazidime KM values are similarly high for both variants (530 μM for KPC-2 and 640 μM for KPC-4), the turnover rate increases 40-fold (kcat 1.9 s−1 for KPC-2 and 81 s−1 for KPC-4). The improved efficiency of ceftazidime hydrolysis by KPC-4 can therefore be attributed to changes in turnover, rather than KM. The two KPC variants also differ in their turnover of other cephalosporins (cefotaxime and nitrocefin). For cefotaxime, a substrate resembling ceftazidime, but with a smaller C7 group (Fig. 1), KM values are lower than those for ceftazidime for both KPC-2 and KPC-4 (200 and 190 μM, respectively) and kcat values substantially faster (76 and 262 s−1), resulting in KPC-4 ~fourfold more efficiently hydrolyzing cefotaxime than KPC-2 (kcat/KM 1.37 μM−1 s−1 compared with 0.38 μM−1 s−1). Conversely, for nitrocefin, a nonclinical reporter substrate (34O'Callaghan C.H. Morris A. Kirby S.M. Shingler A.H. Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate.Antimicrob. Agents Chemother. 1972; 1: 283-288Crossref PubMed Scopus (1420) Google Scholar) similar to earlier cephalosporins, kcat/KM for KPC-2 is tenfold higher than for KPC-4 (34 μM−1 s−1 and 3 μM−1 s−1, respectively) reflecting differences in Michaelis constant (KM = 18 and 59 μM, respectively) and turnover rate (kcat = 610 and 170 s−1, respectively). Thus, compared with KPC-2, the P104R/V240G double substitutions in KPC-4 selectively improve activity against bulkier ESOCs through acceleration of turnover rate (kcat). To further study the basis for KPC activity against the ESOCs cefotaxime and ceftazidime, deacylation-deficient mutants of KPC-2 and KPC-4 were generated by conservative substitution of the general base E166 (35Strynadka N.C.J. Adachi H. Jensen S.E. Johns K. Sielecki A. Betzel C. Sutoh K. James M.N.G. Molecular structure of the acyl-enzyme intermediate in β-lactam hydrolysis at 1.7 Å resolution.Nature. 1992; 359: 700-705Crossref PubMed Scopus (517) Google Scholar) with the isosteric residue Gln (KPC-2E166Q and KPC-4E166Q). Exposure of both KPC-2E166Q and KPC-4E166Q crystals to cephalosporins allowed trapping of the respective acyl-enzymes. We obtained high-resolution crystal structures of the acyl-enzymes of KPC-2E166Q with both ceftazidime and cefotaxime (resolution of 1.25 and 1.31 Å, respectively) and of KPC-4E166Q with ceftazidime (resolution of 1.24 Å). Data collection and refinement statistics are presented in Table 2. Clear Fo − Fc difference density in the KPC active sites allowed confident modeling of the Ser70 acyl-enzymes, with ligand real-space correlation coefficients >0.95 (Fig. S1 and Table S2). Uninterrupted polypeptide chains starting at residue 23 (KPC-2E166Q, KPC-2E166Q:ceftazidime, and KPC-4E166Q:ceftazidime) or 25 (KPC-4E166Q and KPC-2E166Q:cefotaxime) to residue 294 were built into the experimental electron density for all structures except KPC-2E166Q:ceftazidime, for which residues 166 to 172 and 270 to 274 were not well resolved.Table 2Crystallographic data collection and refinement statisticsDatasetKPC-2E166QKPC-2E166Q:cefotaximeKPC-2E166Q:ceftazidimeKPC-4E166QKPC-4E166Q:ceftazidimePDB ID6Z216Z236Z246Z226Z25Data collectionBeamlineDLS I24ALBA XALOC-BL-13DLS I04DLS I04DLS I04Space groupP21212P21212P21212P21212P21212Molecules/ASU11111Cell dimensions a, b, c (Å)60.20, 78.44, 55.9660.14, 78.91, 55.6560.27, 77.21, 55.4160.20, 78.94, 55.9860.23, 77.26, 55.74 α, β, γ (°)90, 90, 9090, 90, 9090, 90, 9090, 90, 9090, 90, 90Resolution (Å)45.56–1.30 (1.32–1.30)45.48–1.31 (1.33–1.31)77.21–1.25 (1.27–1.25)45.66–1.40 (1.42–1.40)55.74–1.24 (1.26–1.24)Rpim0.034 (0.686)0.028 (0.770)0.028 (0.315)0.052 (0.696)0.038 (0.828)CC1/20.999 (0.460)0.999 (0.638)0.999 (0.833)0.999 (0.674)0.999 (0.747)I/σ (I)11.8 (0.9)12.4 (0.9)14.5 (2.2)8.8 (1.3)9.4 (1.0)Completeness (%)100 (100)100 (100)99.2 (98.0)100 (100)98.6 (97.0)Redundancy12.6 (12.2)12.6 (13.1)12.9 (12.4)13.4 (12.3)13.8 (14.3)RefinementResolution (Å)32.86–1.3045.48–1.3155.41–1.2545.66–1.4055.571–1.24No. of reflections65,79364,28771,36853,10373,046Rwork/Rfree0.143/0.1730.1601/0.17920.1497/0.17980.1417/0.17630.1453/0.1704No. of atoms Protein20872133202920632132 Solvent279251285337294 Ligand—5231—62B-factors (Å2) Protein2224162018 Solvent4145373538 Ligand—3217—23RMSD Bond lengths (Å)0.0080.0090.0110.0070.010 Bond angles (°)0.9821.0631.3890.9541.141Ramachandran (%) Outliers0.000.000.000.000.00 Favoured98.998.4898.8198.598.5ASU, asymmetric unit; DLS, Diamond Light Source; ID, identity; KPC, Klebsiella pneumoniae carbapenemase; PDB, Protein Data Bank; RMSD, root-mean-square deviation.Values in parentheses represent information from the highest resolution shells. Open table in a new tab ASU, asymmetric unit; DLS, Diamond Light Source; ID, identity; KPC, Klebsiella pneumoniae carbapenemase; PDB, Protein Data Bank; RMSD, root-mean-square deviation. Values in parentheses represent information from the highest resolution shells. We also determined structures of uncomplexed KPC-2E166Q (resolution of 1.30 Å) and KPC-4E166Q (resolution of 1.40 Å), confirming that mutation at position 166 does not induce substantial changes in the overall fold of the native enzyme (KPC-2 Protein Data Bank [PDB] 5UL8 (33Pemberton O.A. Zhang X. Chen Y. Molecular basis of substrate recognition and product Release by the Klebsiella pneumoniae carbapenemase (KPC-2).J. Med. Chem. 2017; 60: 3525-3530Crossref PubMed Scopus (26) Google Scholar), root-mean-square deviation [RMSD] = 0.157 Å and KPC-4 6QWE (31Tooke C.L. Hinchliffe P. Lang P.A. Mulholland A.J. Brem J. Schofield C.J. Spencer J. Molecular basis of class A β-lactamase inhibition by Relebactam.Antimicrob. Agents Chemother. 2019; 63: e00564-19Crossref PubMed Scopus (20) Google Scholar), RMSD = 0.201 Å for KPC-2E166Q and KPC-4E166Q, respectively; Fig. S2 and Table S3). There are few differences between the active sites of KPC-2 and KPC-2E166Q. The hydrogen bond networks, observed in most class A SBL structures, which involve S70, K73, S130, E166, N170 and an active-site water molecule in the putative deacylating position (labeled DW; Fig. S2 and Table S4), remain unperturbed. In KPC-4, however, a small change in the position of Q166 (compared with E166 in the unmodified enzyme) results in repositioning (by ∼2.4 Å) of the putative deacylating water. The positions of other active-site residues are identical to those observed in native KPC-4 (Fig. S2B and Table S4). Ceftazidime is bound in one clearly resolved conformation in the KPC-2E166Q acyl–enzyme complex and was refined at full occupancy (Fig. S1A). However, in KPC-4E166Q, the 2-carboxypropan-2-yloxyimino group of the ceftazidime C7 substituent (Fig. 1B; teal) was modeled in two conformations: major (A, 75% occupancy) and minor (B, 25% occupancy) in which the orientations of the carboxylate and methyl groups differ by 180° (Fig. S1B and Table S2). Similarly, cefotaxime was refined in complex with KPC-2E166Q in two conformations (A, 49% a
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