A drug-resistant β-lactamase variant changes the conformation of its active-site proton shuttle to alter substrate specificity and inhibitor potency
2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês
10.1074/jbc.ra120.016103
ISSN1083-351X
AutoresVictoria Soeung, Shuo Lu, Liya Hu, Allison Judge, Banumathi Sankaran, B. V. Venkataram Prasad, Timothy Palzkill,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoLys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase–mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, whereas Lys234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a 5-fold increase in kcat for ampicillin, suggesting that the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest that the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest that clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance. Lys234 is one of the residues present in class A β-lactamases that is under selective pressure due to antibiotic use. Located adjacent to proton shuttle residue Ser130, it is suggested to play a role in proton transfer during catalysis of the antibiotics. The mechanism underpinning how substitutions in this position modulate inhibitor efficiency and substrate specificity leading to drug resistance is unclear. The K234R substitution identified in several inhibitor-resistant β-lactamase variants is associated with decreased potency of the inhibitor clavulanic acid, which is used in combination with amoxicillin to overcome β-lactamase–mediated antibiotic resistance. Here we show that for CTX-M-14 β-lactamase, whereas Lys234 is required for hydrolysis of cephalosporins such as cefotaxime, either lysine or arginine is sufficient for hydrolysis of ampicillin. Further, by determining the acylation and deacylation rates for cefotaxime hydrolysis, we show that both rates are fast, and neither is rate-limiting. The K234R substitution causes a 1500-fold decrease in the cefotaxime acylation rate but a 5-fold increase in kcat for ampicillin, suggesting that the K234R enzyme is a good penicillinase but a poor cephalosporinase due to slow acylation. Structural results suggest that the slow acylation by the K234R enzyme is due to a conformational change in Ser130, and this change also leads to decreased inhibition potency of clavulanic acid. Because other inhibitor resistance mutations also act through changes at Ser130 and such changes drastically reduce cephalosporin but not penicillin hydrolysis, we suggest that clavulanic acid paired with an oxyimino-cephalosporin rather than penicillin would impede the evolution of resistance. β-Lactam antibiotics are among the most often used agents for treatment of bacterial infections. Unfortunately, because of their widespread use, bacterial resistance to these drugs now represents a threat to antimicrobial therapy. The major mechanism of resistance to β-lactam antibiotics is the bacterial production of β-lactamases, which hydrolyze the amide bond of the β-lactam ring of these drugs to render them ineffective. β-Lactamases are grouped into four classes, A, B, C, and D, based on amino acid sequence homology (1Ambler R.P. The structure of β-lactamases.Phil. Trans. R. Soc. London B Biol. Sci. 1980; 289 (6109327): 321-33110.1098/rstb.1980.0049Crossref PubMed Scopus (1404) Google Scholar). Enzymes from classes A, C, and D are serine hydrolases that act through a covalent, acyl-enzyme intermediate that is subsequently hydrolyzed by an activated water (2Fisher J.F. Mobashery S. Three decades of the class A β-lactamase acyl-enzyme.Curr. Prot. Pept. Sci. 2009; 10 (19538154): 401-40710.2174/138920309789351967Crossref PubMed Scopus (61) Google Scholar). Enzymes from class B are zinc metalloenzymes where the β-lactam is hydrolyzed through direct attack of an activated water or hydroxide (3Palzkill T. 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In class A enzymes, the catalytic Ser70 residue attacks the carbonyl carbon of the β-lactam, resulting in cleavage of the amide bond to form a covalent acyl-enzyme intermediate (6Strynadka 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 (1436034): 700-70510.1038/359700a-0Crossref PubMed Scopus (529) Google Scholar, 7Escobar W.A. Tan A.K. Fink A.L. Site-directed mutagenesis of β-lactamase leading to accumulation of an acyl-enzyme intermediate.Biochemistry. 1991; 30 (1681903): 10783-1078710.1021/bi00108a02-5Crossref PubMed Scopus (85) Google Scholar). Acylation is facilitated by the transfer of a proton from Ser130, via a proton shuttle from Lys73, to the leaving group nitrogen of the amide (8Nichols D.A. Hargis J.C. Sanishvili R. Jaishankar P. Defrees K. Smith E.W. Wang K.K. Prati F. Renslo A.R. Woodcock H.L. Chen Y. Ligand-induced proton transfer and low-barrier hydrogen bond revealed by X-ray crystallography.J. Am. Chem. Soc. 2015; 137 (26057252): 8086-809510.1021/jacs.5b00749Crossref PubMed Scopus (59) Google Scholar, 9Pemberton O.A. Noor R.E. Vasantha Kumar M.V. Sanishvili R. Kemp M.T. Kearns F.L. Woodcock H.L. Gelis I. Chen Y. Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam.Proc. Natl. Acad. Sci. U. S. A. 2020; 117 (32123084): 5818-582510.1073/pnas.1922203117Crossref PubMed Scopus (22) Google Scholar) (Fig. 1). The deacylation reaction is catalyzed by the active-site residue Glu166, which serves as a general base to activate a catalytic water molecule for attack on the carbonyl carbon of the acyl-intermediate and subsequent formation of the hydrolyzed product. A minimal kinetic scheme for β-lactamase–catalyzed hydrolysis of β-lactams is shown in Fig. 2A. These active-site residues are conserved across all class A β-lactamases, consistent with their key role in catalysis.Figure 2Kinetic model for serine active-site β-lactamases. A, at the top is the minimal scheme for the reaction with E as free enzyme, S as substrate, ES as enzyme-substrate complex, EAc as acyl-enzyme intermediate, and P as product. Also shown are the equations for kcat, Km, and kcat/Km based on the kinetic scheme. A simplified model is shown at the right based on the assumption that k−1 ⋙ k2 (i.e. that the substrate-binding reaction is in rapid equilibrium). B, schematic illustration of the β-lactamase mechanism for penicillin hydrolysis with Ser70 serving as the nucleophile for attack on the β-lactam carbonyl carbon to form the acyl-enzyme intermediate. Subsequently, Glu-166 acts as a base to activate water for attack on the carbonyl carbon of the acyl-enzyme intermediate, leading to hydrolyzed product. C, schematic illustration of the mechanism for cephalosporin hydrolysis. Note that the R2 group is eliminated. The elimination of the R2 group is shown coincident with cleavage of the β-lactam amide bond, although elimination can also occur after cleavage of the amide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In response to increasing antibiotic resistance due to the production of β-lactamases, extended-spectrum oxyimino-cephalosporins were introduced in the 1980s. These antibiotics are poor substrates for widespread class A β-lactamases, such as TEM-1 and SHV-1, and therefore bacteria containing these enzymes remain susceptible to the drugs. The use of oxyimino-cephalosporins, however, led to selective pressure for the evolution of resistance, resulting in mutations in TEM and SHV that expand their substrate specificity (10Palzkill T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases.Front. Mol. Biosci. 2018; 5 (29527530): 1610.3389/fmolb.2018.00016Crossref PubMed Scopus (94) Google Scholar). β-Lactamase inhibitors have also been developed to combat antibiotic resistance (11Drawz S.M. Bonomo R.A. Three decades of β-lactamase inhibitors.Clin. Microb. Rev. 2010; 23 (20065329): 160-20110.1128/CMR.00037-09Crossref PubMed Scopus (1185) Google Scholar). Clavulanic acid, tazobactam, and sulbactam are covalent inhibitors of many class A β-lactamases. They act through secondary reactions that occur after acylation due to secondary ring opening and fragmentation that attaches various fragments to Ser70 as well as one product that is cross-linked to Ser130 (11Drawz S.M. Bonomo R.A. Three decades of β-lactamase inhibitors.Clin. Microb. Rev. 2010; 23 (20065329): 160-20110.1128/CMR.00037-09Crossref PubMed Scopus (1185) Google Scholar). 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More recently, the diazabicyclooctane, avibactam, has been introduced, which is also a covalent inhibitor that cycles between acylation and deacylation without secondary ring fragmentation (17Ehmann D.E. Jahic H. Ross P.L. Gu R.-G. Hu J. Durand-Réville T.F. Lahiri S. Thresher J. Livchak S. Gao N. Palmer T. Walkup G.K. Fisher S.L. Kinetics of avibactam inhibition against class A, C, and D β-lactamases.J. Biol. Chem. 2013; 288 (23913691): 27960-2797110.1074/jbc.M113.485979Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 18Bush 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 (231) Google Scholar). The selective pressure of oxyimino-cephalosporin use has also led to the emergence of a new family of β-lactamases that readily hydrolyze these drugs. The CTX-M family first appeared in the late 1980s and now represents the most widespread extended-spectrum β-lactamases in Gram-negative bacteria. The CTX-M β-lactamases are class A enzymes that have a broad substrate specificity that includes penicillins and cephalosporins, including the oxyimino-cephalosporin cefotaxime (10Palzkill T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases.Front. Mol. Biosci. 2018; 5 (29527530): 1610.3389/fmolb.2018.00016Crossref PubMed Scopus (94) Google Scholar). They are divided into five clusters based on amino acid sequence homology, including CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25, with the names based on the prominent member of each subgroup (19D'Andrea M.M. Arena F. Pallecchi L. Rossolini G.M. CTX-M-type β-lactamases: a successful story of antibiotic resistance.Int. J. Med. Microbiol. 2013; 303 (23490927): 305-31710.1016/j.ijmm.2013.02.008Crossref PubMed Scopus (303) Google Scholar). The subgroups differ from one another by ≥10% amino acid sequence divergence, and each subgroup contains variants that differ by ≤5% sequence divergence (19D'Andrea M.M. Arena F. Pallecchi L. Rossolini G.M. CTX-M-type β-lactamases: a successful story of antibiotic resistance.Int. J. Med. Microbiol. 2013; 303 (23490927): 305-31710.1016/j.ijmm.2013.02.008Crossref PubMed Scopus (303) Google Scholar). The substrate specificity of β-lactamases is a key determinant of antibiotic resistance in that it determines what drugs are hydrolyzed and therefore the susceptibility of bacteria harboring the enzymes. Most class A β-lactamases efficiently hydrolyze penicillins and cephalosporins to provide bacterial resistance to these drugs. For example, the common TEM-1 β-lactamase hydrolyzes benzylpenicillin with a catalytic efficiency in the range of 107–108m−1 s−1 and the older cephalosporin, cephalothin, with a kcat/Km of 3 × 105m−1 s−1, whereas it poorly hydrolyzes the oxyimino-cephalosporin cefotaxime, with a kcat/Km of 2 × 103m−1 s−1 (10Palzkill T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases.Front. Mol. Biosci. 2018; 5 (29527530): 1610.3389/fmolb.2018.00016Crossref PubMed Scopus (94) Google Scholar, 20Christensen H. Martin M. Waley G. β-Lactamases as fully efficient enzymes: determination of all the rate constants in the acyl-enzyme mechanism.Biochem. J. 1990; 266 (2158301): 853-861PubMed Google Scholar). In contrast, CTX-M β-lactamases hydrolyze benzylpenicillin with a kcat/Km of 5 × 106m−1 s−1, cephalothin with a kcat/Km of 2 × 107m−1 s−1, and cefotaxime with a kcat/Km of 3 × 106m−1 s−1 (10Palzkill T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases.Front. Mol. Biosci. 2018; 5 (29527530): 1610.3389/fmolb.2018.00016Crossref PubMed Scopus (94) Google Scholar, 21Adamski C.J. Cardenas A.M. Brown N.G. Horton L.B. Sankaran B. Prasad B.V. Gilbert H.F. Palzkill T. Molecular basis for the catalytic specificity of the CTX-M extended spectrum β-lactamases.Biochemistry. 2015; 54 (25489790): 447-45710.1021/bi501195gCrossref PubMed Scopus (43) Google Scholar). Thus, both TEM-1 and CTX-M enzymes efficiently catalyze the hydrolysis of penicillins and early-generation cephalosporins, but CTX-M also hydrolyzes the newer, oxyimino-cephalosporin cefotaxime ∼1,000-fold faster than the TEM-1 enzyme (10Palzkill T. Structural and mechanistic basis for extended-spectrum drug-resistance mutations in altering the specificity of TEM, CTX-M, and KPC β-lactamases.Front. Mol. Biosci. 2018; 5 (29527530): 1610.3389/fmolb.2018.00016Crossref PubMed Scopus (94) Google Scholar). This enhanced hydrolysis of cefotaxime provides resistance to bacteria containing CTX-M enzymes and has led to the rapid spread of CTX-M–encoding plasmids among pathogens (19D'Andrea M.M. Arena F. Pallecchi L. Rossolini G.M. CTX-M-type β-lactamases: a successful story of antibiotic resistance.Int. J. Med. Microbiol. 2013; 303 (23490927): 305-31710.1016/j.ijmm.2013.02.008Crossref PubMed Scopus (303) Google Scholar). Another active-site residue that is conserved across most class A enzymes and among the serine β-lactamases in general is Lys234 (Fig. 1D). Based on structures of β-lactamases in complex with β-lactam antibiotics, Lys234 has been suggested to facilitate substrate binding by providing an electrostatic environment favorable for interaction with the C3/C4 carboxylate group found in all of the β-lactams (6Strynadka 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 (1436034): 700-70510.1038/359700a-0Crossref PubMed Scopus (529) Google Scholar, 22Herzberg O. Moult J. Bacterial resistance to β-lactam antibiotics: crystal structure of β-lactamase from Staphylococcus aureus PC1 at 2.5 Å resolution.Science. 1987; 236 (3107125): 694-70110.1126/science.3107125Crossref PubMed Scopus (290) Google Scholar, 23Chen Y. Bonnet R. Shoichet B.K. The acylation mechanism of CTX-M β-lactamase at 0.88 Å resolution.J. Am. Chem. Soc. 2007; 129 (17408273): 5378-538010.1021/ja0712064Crossref PubMed Scopus (56) Google Scholar) (Fig. 3). In addition, Lys234 is located near the proton shuttle residue, Ser130, and may act to lower the pKa of the serine hydroxyl to facilitate proton transfer to the nitrogen leaving group of the β-lactam during the acylation reaction (8Nichols D.A. Hargis J.C. Sanishvili R. Jaishankar P. Defrees K. Smith E.W. Wang K.K. Prati F. Renslo A.R. Woodcock H.L. Chen Y. Ligand-induced proton transfer and low-barrier hydrogen bond revealed by X-ray crystallography.J. Am. Chem. Soc. 2015; 137 (26057252): 8086-809510.1021/jacs.5b00749Crossref PubMed Scopus (59) Google Scholar, 9Pemberton O.A. Noor R.E. Vasantha Kumar M.V. Sanishvili R. Kemp M.T. Kearns F.L. Woodcock H.L. Gelis I. Chen Y. Mechanism of proton transfer in class A β-lactamase catalysis and inhibition by avibactam.Proc. Natl. Acad. Sci. U. S. A. 2020; 117 (32123084): 5818-582510.1073/pnas.1922203117Crossref PubMed Scopus (22) Google Scholar) (Fig. 1). Studies with TEM-1 β-lactamase have shown that a K234T substitution that removes the positive charge results in greatly reduced (∼50-fold) kcat values and 50-fold increased Km values for benzylpenicillin hydrolysis, suggesting that Lys234 is important for both substrate binding and catalysis (24Lenfant F. Labia R. Masson J.-M. Replacement of lysine 234 affects transition state stablization in the active site of β-lactamase TEM1.J. Biol. Chem. 1991; 266: 17187-17194Abstract Full Text PDF PubMed Google Scholar). In addition, the K234T substitution essentially eliminates cephalosporin hydrolysis by the TEM-1 enzyme (24Lenfant F. Labia R. Masson J.-M. Replacement of lysine 234 affects transition state stablization in the active site of β-lactamase TEM1.J. Biol. Chem. 1991; 266: 17187-17194Abstract Full Text PDF PubMed Google Scholar). In contrast, a K234R substitution in TEM-1 has relatively modest effects on penicillin hydrolysis (kcat reduced 2-fold) and no effect on the rate of cephalosporin hydrolysis (24Lenfant F. Labia R. Masson J.-M. Replacement of lysine 234 affects transition state stablization in the active site of β-lactamase TEM1.J. Biol. Chem. 1991; 266: 17187-17194Abstract Full Text PDF PubMed Google Scholar). Taken together, these results suggest that Lys234 may contribute to substrate binding, through interaction with the C3/C4 carboxylate group, and to catalysis, by lowering the pKa of Ser130. There is also evidence that Lys234 has a role in determining the substrate specificity and inhibitor susceptibility of class A β-lactamases. Although lysine is conserved at position 234 among most class A enzymes, this residue is an arginine in a subclass of enzymes named carbenicillinases that includes the Pseudomonas aeruginosa PSE-4 β-lactamase (25Lim D. Sanschagrin F. Passmore L. De Castro L. Levesque R.C. Strynadka N.C. Insights into the molecular basis for the carbenicillinase activity of PSE-4 β-lactamase from crystallographic and kinetic studies.Biochemistry. 2001; 40 (11148033): 395-40210.1021/bi001653vCrossref PubMed Scopus (38) Google Scholar). These enzymes are characterized by the ability to efficiently hydrolyze carbenicillin, and thus, they exhibit an altered substrate specificity compared with the canonical TEM-1 β-lactamase (25Lim D. Sanschagrin F. Passmore L. De Castro L. Levesque R.C. Strynadka N.C. Insights into the molecular basis for the carbenicillinase activity of PSE-4 β-lactamase from crystallographic and kinetic studies.Biochemistry. 2001; 40 (11148033): 395-40210.1021/bi001653vCrossref PubMed Scopus (38) Google Scholar, 26Philippon A. Slama P. Deny P. Labia R. A structure-based classification of class A β-lactamases, a broadly diverse family of enzymes.Clin. Microb. Rev. 2016; 29 (26511485): 29-5710.1128/CMR.00019-15Crossref PubMed Scopus (74) Google Scholar). In addition, the natural variants SHV-72 and SHV-84 contain the K234R substitution, and these enzymes display reduced susceptibility to the β-lactamase inhibitor clavulanic acid and lower catalytic efficiency for cephalosporin hydrolysis (15Mendonça N. Manageiro V. Robin F. Salgado M.J. Ferreira E. Caniça M. Bonnet R. The Lys234Arg substitution in the enzyme SHV-72 is a determinant for resistance to clavulanic acid inhibition.Antimicrob. Agents Chemother. 2008; 52 (18316518): 1806-181110.1128/AAC.01381-07Crossref PubMed Scopus (23) Google Scholar, 27Manageiro V. Ferreira E. Albuquerque L. Bonnet R. Caniça M. Biochemical study of a new inhibitor-resistant beta-lactamase, SHV-84, produced by a clinical Escherichia coli strain.Antimicrob. Agents Chemother. 2010; 54 (20211886): 2271-227210.1128/AAC.01442-09Crossref PubMed Scopus (11) Google Scholar). Finally, the CTX-M-106 and -107 natural variants contain the K234R substitution, and selection of CTX-M-14 mutants for increased resistance to clavulanic acid led to the isolation of a K234R mutant (19D'Andrea M.M. Arena F. Pallecchi L. Rossolini G.M. CTX-M-type β-lactamases: a successful story of antibiotic resistance.Int. J. Med. Microbiol. 2013; 303 (23490927): 305-31710.1016/j.ijmm.2013.02.008Crossref PubMed Scopus (303) Google Scholar, 28Ripoll A. Baquero F. Novais A. Rodríguez-Domínguez M.J. Turrientes M.-C. Cantón R. Galán J.-C. In vitro selection of variants resistant to β-lactams plus β-lactamase inhibitors in CTX-M β-lactamases: predicting the in vivo scenario?.Antimicrob. Agents Chemother. 2011; 55 (21788458): 4530-453610.1128/AAC.00178-11Crossref PubMed Scopus (37) Google Scholar). Subsequent characterization of the enzyme revealed decreased susceptibility to clavulanic acid and decreased hydrolysis of cephalosporins (28Ripoll A. Baquero F. Novais A. Rodríguez-Domínguez M.J. Turrientes M.-C. Cantón R. Galán J.-C. In vitro selection of variants resistant to β-lactams plus β-lactamase inhibitors in CTX-M β-lactamases: predicting the in vivo scenario?.Antimicrob. Agents Chemother. 2011; 55 (21788458): 4530-453610.1128/AAC.00178-11Crossref PubMed Scopus (37) Google Scholar). To gain a mechanistic understanding of how Lys234 and its various substitutions differentially alter CTX-M β-lactamase substrate specificity and the associated resistance profile, we examined the sequence requirements at position 234 for penicillin and cephalosporin hydrolysis using codon randomization mutagenesis, which revealed that lysine is required for cephalosporin hydrolysis, whereas either lysine or arginine is consistent with penicillin hydrolysis. Subsequent purification of the K234R enzyme and kinetic analysis showed that the catalytic efficiency for ampicillin hydrolysis was unchanged, whereas kcat/Km was 100-fold lower for cefotaxime hydrolysis. In addition, kinetics experiments revealed a 1,500-fold reduced acylation rate for the K234R enzyme, consistent with disruption of the Ser130-mediated protonation of the β-lactam leaving group nitrogen. Further, X-ray structures of the K234R apoenzyme and complexes with ampicillin and cefotaxime revealed an altered conformation of Ser130, again consistent with a defect in acylation. Finally, we show that clavulanic acid is a less potent inhibitor of the K234R enzyme and that the change in conformation of Ser130 is correlated with the loss of inhibitor potency. These findings suggest that clavulanic acid should be paired with an oxyimino-cephalosporin rather than a penicillin to slow the evolution of resistance because mutations that alter the presence or position of Ser130 block cephalosporin hydrolysis. To examine the role of Lys234 in CTX-M enzyme catalysis and substrate specificity, we determined the amino acid sequence requirements at position 234 for penicillin and cephalosporin hydrolysis. We reasoned that, if the sequence requirements are similar for each substrate, the position is not a determinant of substrate specificity (i.e. substitutions do not differentially affect penicillin versus cephalosporin hydrolysis) (29Cantu C. Huang W. Palzkill T. Cephalosporin substrate specificity determinants of TEM-1 β-lactamase.J. Biol. Chem. 1997; 272 (9360991): 29144-2915010.1074/jbc.272.46.2914-4Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). For this purpose, we used site-directed mutagenesis to randomize the DNA sequence of the codon to create a library of all possible substitutions at the position (30Sun Z. Hu L. Sankaran B. Prasad B.V.V. Palzkill T. Differential active site requirements for NDM-1 β-lactamase hydrolysis of carbapenem versus penicillin and cephalosporin antibiotics.Nat. Commun. 2018; 9 (30375382)452410.1038/s41467-018-06839-1Crossref PubMed Scopus (47) Google Scholar). The library was introduced into Escherichia coli, and the resultant colonies were pooled and grown in media containing high concentrations of ampicillin or cefotaxime that selected for high enzyme activity as well as low concentrations where only partial activity was required for bacteria to survive the selection (Figure 3, Figure 4). Plasmid DNA was recovered from each culture after overnight growth under selective conditions, and the region of the bla gene containing the residue 234 region was amplified by PCR with a 7-bp barcode at the 5′-end of each primer to uniquely identify each experiment. The PCR products were pooled, and next-generation sequencing (NGS) was performed to determine the spectrum of amino acids that are consistent with CTX-M-14 function under each selective condition. The deep-sequencing results revealed that the naive library without antibiotic selection was diverse and all amino acids were present at similar frequencies (Fig. 4A). In contrast, arginine and lysine were significantly enriched after selection in both high- and low-ampicillin conditions, whereas all other amino acids were present at low frequency (Fig. 4B). The deep-sequencing results for the low-concentration cefotaxime selection showed that lysine and arginine were enriched but not to the extent observed for the ampicillin selections, as other amino acids were present at significant frequencies (Fig. 4A). At high cefotaxime concentrations, however, lysine was dominant and present at a much higher frequency than other amino acids, including arginine (Fig. 4B). Taken together, these results suggest that either lysine or arginine at position 234 supports high levels of ampicillin resistance and, presumably, high catalytic efficiency for ampicillin hydrolysis. In contrast, only lysine appears to provide high-level resistance and high levels of enzyme activity toward cefotaxime. Therefore, the identity of the amino acid at position 234 strongly influences the penicillinase versus cephalosporinase characteristics of the CTX-M enzyme. The dominance of lysine in random library selections and subsequent deep-sequencing results at high cefotaxime concentrations predicts that the CTX-M-14 K234R-substituted enzyme should display high catalytic efficiency for ampicillin hydrolysis but reduced activity toward cefotaxime. To test this hypothesis, we purified the K234R enzyme and performed steady-state kinetic analysis with ampicillin and cefotaxime as substrates (Fig. 3 and Table 1). The kcat for ampicillin hydrolysis for the K234R enzyme is increased 5-fold and Km is increased 9-fold, whereas kcat/Km is only modestly changed compared with the WT enzyme (Table 1). With cefotaxime as substrate, kcat for the K234R enzyme is drastically reduced (500-fold), whereas Km is reduced 6-fold and kcat/Km is r
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