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Robert A Bonomo - One of the best experts on this subject based on the ideXlab platform.
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inactivation of the pseudomonas derived Cephalosporinase 3 pdc 3 by relebactam
Antimicrobial Agents and Chemotherapy, 2018Co-Authors: Christopher R Bethel, Melissa D Barnes, Jim Alsop, Scott A Becka, Joseph D Rutter, Krisztina M Pappwallace, Robert A BonomoAbstract:Pseudomonas aeruginosa is a prevalent and life-threatening Gram-negative pathogen. Pseudomonas-derived cephlosporinase (PDC) is the major inducible Cephalosporinase in P. aeruginosa In this investigation, we show that relebactam, a diazabicyclooctane β-lactamase inhibitor, potently inactivates PDC-3, with a k2/K of 41,400 M-1 s-1 and a koff of 0.00095 s-1 Relebactam restored susceptibility to imipenem in 62% of multidrug-resistant P. aeruginosa clinical isolates, while only 21% of isolates were susceptible to imipenem-cilastatin alone. Relebactam promises to increase the efficacy of imipenem-cilastatin against P. aeruginosa.
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structure based analysis of boronic acids as inhibitors of acinetobacter derived Cephalosporinase 7 a unique class c β lactamase
ACS Infectious Diseases, 2018Co-Authors: Alexandra A Bouza, Robert A Bonomo, Hollister C Swanson, Magdalena A Taracila, Chiara Romagnoli, Emilia Caselli, Fabio Prati, Kali A Smolen, Alison L VandineAbstract:Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to β-lactam antibiotics in Acinetobacter spp. is mediated by class C β-lactamases known as Acinetobacter-derived Cephalosporinases (ADCs). ADCs are unaffected by clinically used β-lactam-based β-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C Cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Ki values ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the β-lactamase–inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 A). In the ADC-7/CR192 complex, the BATSI with the lowest Ki (0.45 nM) and greatest ΔTm (+9 °C), a trifluoromethyl substituent, interacts ...
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biochemical and structural analysis of inhibitors targeting the adc 7 Cephalosporinase of acinetobacter baumannii
Biochemistry, 2014Co-Authors: Rachel A Powers, Robert A Bonomo, Hollister C Swanson, Magdalena A Taracila, Nicholas W Florek, Chiara Romagnoli, Emilia Caselli, Fabio PratiAbstract:β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived Cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C Cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal Cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 A resolution) and in complex with S02030 (2.0 A resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030...
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multicenter evaluation of a new dna microarray for rapid detection of clinically relevant bla genes from beta lactam resistant gram negative bacteria
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Pierre Bogaerts, Thierry Naas, Patrice Nordmann, Andrea M Hujer, Roberta Rezende De Castro, Andrea Endimiani, Youri Glupczynski, Robert A BonomoAbstract:A new commercial low-density microarray which identifies common extended-spectrum β-lactamase plasmid-mediated Cephalosporinase genes, as well as carbapenemase (bla(KPC) and bla(NDM)) genes, was evaluated. We tested 207 clinical and reference/collection isolates of the Enterobacteriaceae possessing different bla genes. Overall, the sensitivity and specificity of the microarray were 100% for the detection of the plasmid-mediated bla(AmpC), bla(KPC), and bla(NDM) genes using bla gene sequencing as the reference method.
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exploring sequence requirements for c3 c4 carboxylate recognition in the pseudomonas aeruginosa Cephalosporinase insights into plasticity of the ampc β lactamase
Protein Science, 2011Co-Authors: Sarah M Drawz, Emilia Caselli, Magdalena A Taracila, Fabio Prati, Robert A BonomoAbstract:In Pseudomonas aeruginosa, the chromosomally encoded class C Cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived Cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C3/C4 carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C3/C4 carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C3/C4 β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C3/C4 β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C3/C4 carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.
Patrice Nordmann - One of the best experts on this subject based on the ideXlab platform.
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multicenter evaluation of a new dna microarray for rapid detection of clinically relevant bla genes from beta lactam resistant gram negative bacteria
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Pierre Bogaerts, Thierry Naas, Patrice Nordmann, Andrea M Hujer, Roberta Rezende De Castro, Andrea Endimiani, Youri Glupczynski, Robert A BonomoAbstract:A new commercial low-density microarray which identifies common extended-spectrum β-lactamase plasmid-mediated Cephalosporinase genes, as well as carbapenemase (bla(KPC) and bla(NDM)) genes, was evaluated. We tested 207 clinical and reference/collection isolates of the Enterobacteriaceae possessing different bla genes. Overall, the sensitivity and specificity of the microarray were 100% for the detection of the plasmid-mediated bla(AmpC), bla(KPC), and bla(NDM) genes using bla gene sequencing as the reference method.
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extended spectrum Cephalosporinase in acinetobacter baumannii
Antimicrobial Agents and Chemotherapy, 2010Co-Authors: Josemanuel Rodriguezmartinez, Patrice Nordmann, Esthel Ronco, Laurent PoirelAbstract:An AmpC-type beta-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C beta-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Omega-loop) compared to a reference AmpC Cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.
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detection of a plasmid mediated inducible Cephalosporinase dha 1 from escherichia coli
Pathology, 2010Co-Authors: Jeannette N Pham, Laurent Poirel, Patrice Nordmann, I Chambers, S M BellAbstract:Sir,Ambler class C Cephalosporinases (AmpC) are produced by members of the Enterobacteriaceae that carry a chromosomal ampC gene such as Enterobacter cloacae, Serratia marcescens and Morganella mor...
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extended spectrum Cephalosporinases structure detection and epidemiology
Future Microbiology, 2007Co-Authors: Patrice Nordmann, Hedi MammeriAbstract:Extended-spectrum AmpC β-lactamases of Enterobacteriaceae, which are chromosomally or plasmid-encoded, possess structural modifications in the vicinity of the active site compared with their progenitors. They display an increased catalytic efficiency against extended-spectrum β-lactams, such as ceftazidime, cefotaxime, cefepime, cefpirome and, in some cases, also against imipenem. An overview of the molecular and biochemical characterization of this recently identified mechanism of resistance to β-lactams is provided as well as its prevalence and possible clinical significance.
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ertapenem resistance of escherichia coli
Emerging Infectious Diseases, 2007Co-Authors: Mariefrederique Lartigue, Laurent Poirel, Claire Poyart, Helene Reglierpoupet, Patrice NordmannAbstract:Of all β-lactam antimicrobial drugs, carbapenems (imipenem, meropenem, and ertapenem) have the most consistent activity against Enterobacteriaceae. Activity is retained against most isolates that produce high-level AmpC β-lactamases (Cephalosporinases) and clavulanic-acid–inhibited extended-spectrum β-lactamases (ESBL) (1). However, a few carbapenem-resistant enterobacterial isolates have been reported; resistance may be caused by production of carbapenemases (2) or by combined mechanisms of an outer membrane permeability defect and extended-spectrum β-lactamases or Cephalosporinase (3–6). Spread of CTX-M type ESBLs, especially in Escherichia coli, may provide a favorable background for selection of carbapenem resistance. Resistance to the recently introduced ertapenem has not been reported in E. coli associated with a CTX-M-type enzyme. We describe the clinical and microbiologic features associated with an ertapenem-resistant E. coli isolate that had reduced susceptibility to imipenem after in vivo treatment with imipenem/cilastatin and provide a detailed molecular analysis of the antimicrobial drug resistance mechanisms.
Samarendra N. Maiti - One of the best experts on this subject based on the ideXlab platform.
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synthesis and biological evaluation of penam sulfones as inhibitors of β lactamases
Bioorganic & Medicinal Chemistry, 2005Co-Authors: Oludotun A. Phillips, R. G. Micetich, David Czajkowski, Paul Spevak, Andhe Narender V Reddy, Eduardo Setti, Herninder Atwal, Sameeh Salama, Samarendra N. MaitiAbstract:The chemical synthesis of a series of new penam sulfone derivatives bearing a 2beta-substituted-oxyimino and -hydrazone substituents, their beta-lactamase inhibitory properties against selected enzymes representing class A and C beta-lactamases are reported. The oxime containing penam sulfones strongly inhibited the Escherichia coli TEM-1 and Klebsiella pneumoniae cefotaximase (CTX-1) enzymes, but moderately inhibited the Pseudomonas aeruginosa 46012 Cephalosporinase; while the 2beta-substituted-hydrazone derivatives were generally less active against these enzymes. Furthermore, most of the inhibitors enhanced the antibacterial activities of piperacillin (PIP) and ceftazidime (CAZ) particularly against TEM-1 and CTX-1 producing bacterial strains.
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studies on penam sulfones iii synthesis and β lactamase inhibitory activity of sodium 6r 6 α hydroxybenzyl 2β methoxyiminomethyl 2α methylpenam 3α carboxylate 1 1 dioxide and sodium 2β acyl 2α methylpenam 3α carboxylate 1 1 dioxide
Bioorganic & Medicinal Chemistry Letters, 1997Co-Authors: David Czajkowski, Chieko Kunugita, R. G. Micetich, Oludotun A. Phillips, Andhe Narender V Reddy, Eduardo Setti, Samarendra N. MaitiAbstract:Abstract The synthesis and in vitro synergies of (6R)-6-(α-hydroxybenzyl)-2β-methoxyiminomethyl-2α-methylpenam-3α-carboxylate 1,1-dioxide ( 4 ) and 2β-acyl-2α-methylpenam-3α-carboxylate 1,1-dioxide ( 15 ) are described. Compound 15 showed good in vitro synergy in combination with piperacillin and ceftazidime against chromosomally mediated class I Cephalosporinase producing organisms including TEM, SHV, and OXA-type enzymes producing microorganisms.
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chemical modification of tazobactam synthesis of 2 beta 4 substituted 1 2 3 triazol 1 yl methyl penicillanic acid sulfone derivatives
The Journal of Antibiotics, 1995Co-Authors: Eduardo L Setti, Charles Fiakpui, David P Czajkowski, Chieko Kunugita, Samarendra N. Maiti, R. G. Micetich, Oludotun A. Phillips, Kevin Atchison, Akio HyodoAbstract:A series of 2β-[(4-substituted)-l, 2, 3-triazol-l-yl]methyl penicillanic acid sulfones was synthesized as β-lactamase inhibitors. Many of these compounds showed good in vitro inhibitory activity against penicillinase, cefotaximase and plasmid-mediated class III TEM enzymes, but exhibited weaker Cephalosporinase inhibition. One member in this series-2β-[(4-pyridiniummethyl)-l, 2, 3-triazol-lyl]methyl-6, 6-dihydropenicillanate 1, 1-dioxide (12a), when tested in combination with piperacillin, showed excellent synergistic activity against microorganisms producing plasmid-mediated enzymes, but had insufficient activity against microorganisms producing chromosomally mediated class I enzymes.
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synthesis and β lactamase inhibitory activity of 6 1 heteroarylthioethyl 1 2 3 triazol 4 yl methylene penam sulfones
ChemInform, 1995Co-Authors: Samarendra N. Maiti, R. G. Micetich, Oludotun A. Phillips, Kevin Atchison, Mohsen Daneshtalab, Chieko KunugitaAbstract:The synthesis of beta-lactamase inhibitory activity of a series of sodium 6-[(1-heteroarylthioethyl-1,2,3-triazol-4-yl)methylene]pe nicillanate, 1,1-dioxides are described. Their activity was compared with tazobactam and sulbactam. The Z-isomers were more active than the E-isomers. The in vitro activity of the Z-isomers of the phenylthiadiazole derivatives (13a and 15a) was better than sulbactam against the tested beta-lactamases and comparable to tazobactam especially against TEM-2 and Cephalosporinase. But their synergistic activity with five antibiotics was inferior to tazobactam.
Laurent Poirel - One of the best experts on this subject based on the ideXlab platform.
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extended spectrum Cephalosporinase in acinetobacter baumannii
Antimicrobial Agents and Chemotherapy, 2010Co-Authors: Josemanuel Rodriguezmartinez, Patrice Nordmann, Esthel Ronco, Laurent PoirelAbstract:An AmpC-type beta-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C beta-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Omega-loop) compared to a reference AmpC Cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.
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detection of a plasmid mediated inducible Cephalosporinase dha 1 from escherichia coli
Pathology, 2010Co-Authors: Jeannette N Pham, Laurent Poirel, Patrice Nordmann, I Chambers, S M BellAbstract:Sir,Ambler class C Cephalosporinases (AmpC) are produced by members of the Enterobacteriaceae that carry a chromosomal ampC gene such as Enterobacter cloacae, Serratia marcescens and Morganella mor...
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ertapenem resistance of escherichia coli
Emerging Infectious Diseases, 2007Co-Authors: Mariefrederique Lartigue, Laurent Poirel, Claire Poyart, Helene Reglierpoupet, Patrice NordmannAbstract:Of all β-lactam antimicrobial drugs, carbapenems (imipenem, meropenem, and ertapenem) have the most consistent activity against Enterobacteriaceae. Activity is retained against most isolates that produce high-level AmpC β-lactamases (Cephalosporinases) and clavulanic-acid–inhibited extended-spectrum β-lactamases (ESBL) (1). However, a few carbapenem-resistant enterobacterial isolates have been reported; resistance may be caused by production of carbapenemases (2) or by combined mechanisms of an outer membrane permeability defect and extended-spectrum β-lactamases or Cephalosporinase (3–6). Spread of CTX-M type ESBLs, especially in Escherichia coli, may provide a favorable background for selection of carbapenem resistance. Resistance to the recently introduced ertapenem has not been reported in E. coli associated with a CTX-M-type enzyme. We describe the clinical and microbiologic features associated with an ertapenem-resistant E. coli isolate that had reduced susceptibility to imipenem after in vivo treatment with imipenem/cilastatin and provide a detailed molecular analysis of the antimicrobial drug resistance mechanisms.
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plasmid mediated and inducible Cephalosporinase dha 2 from klebsiella pneumoniae
Journal of Antimicrobial Chemotherapy, 2001Co-Authors: Nicolas Fortineau, Laurent Poirel, Patrice NordmannAbstract:A Klebsiella pneumoniae strain resistant to cefoxitin and oxyimino-cephalosporins was cultured from a child hospitalized in Paris, France, in 1992. This isolate harboured a beta-lactamase gene located on an approximately 200 kb non-self-transferable plasmid. The beta-lactamase identified, DHA-2, shared 99% amino acid identity with the AmpC enzyme of Morganella morganii. DHA-2 was a point-mutant derivative of DHA-1 identified previously in a Salmonella enteritidis isolate. DHA-2 expression was inducible due to an ampR regulatory gene. This is the first report of an inducible and plasmid-located Cephalosporinase from K. pneumoniae.
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biochemical genetic characterization and regulation of expression of an acc 1 like chromosome borne Cephalosporinase from hafnia alvei
Antimicrobial Agents and Chemotherapy, 2000Co-Authors: Delphine Girlich, Laurent Poirel, Thierry Naas, Samuel Bellais, Amal Karim, Patrice NordmannAbstract:A naturally occurring AmpC β-lactamase (Cephalosporinase) gene was cloned from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coli. The deduced AmpC β-lactamase (ACC-2) had a pI of 8 and a relative molecular mass of 37 kDa and showed 50 and 47% amino acid identity with the chromosome-encoded AmpCs from Serratia marcescens and Providentia stuartii, respectively. It had 94% amino acid identity with the recently described plasmid-borne Cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting the chromosomal origin of ACC-1. The hydrolysis constants (kcat and Km) showed that ACC-2 was a peculiar Cephalosporinase, since it significantly hydrolyzed cefpirome. Once its gene was cloned and expressed in E. coli (pDEL-1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an uncommon reduced susceptibility to cefpirome. A divergently transcribed ampR gene with an overlapping promoter compared with ampC (blaACC-2) was identified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid identity with the closest AmpR protein from P. stuartii. β-Lactamase induction experiments showed that the ampC gene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H. alvei 1 cultures that expressed an inducible-Cephalosporinase phenotype, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plates, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced Cephalosporinase. Transformation of H. alvei 1 DER or E. coli JRG582 (ampDE mutant) harboring ampC and ampR from H. alvei 1 with a recombinant plasmid containing ampD from E. coli resulted in a decrease in the MIC of β-lactam and recovery of an inducible phenotype for H. alvei 1 DER. Thus, AmpR and AmpD proteins may regulate biosynthesis of the H. alvei Cephalosporinase similarly to other enterobacterial Cephalosporinases.
Magdalena A Taracila - One of the best experts on this subject based on the ideXlab platform.
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structure based analysis of boronic acids as inhibitors of acinetobacter derived Cephalosporinase 7 a unique class c β lactamase
ACS Infectious Diseases, 2018Co-Authors: Alexandra A Bouza, Robert A Bonomo, Hollister C Swanson, Magdalena A Taracila, Chiara Romagnoli, Emilia Caselli, Fabio Prati, Kali A Smolen, Alison L VandineAbstract:Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to β-lactam antibiotics in Acinetobacter spp. is mediated by class C β-lactamases known as Acinetobacter-derived Cephalosporinases (ADCs). ADCs are unaffected by clinically used β-lactam-based β-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C Cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Ki values ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the β-lactamase–inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 A). In the ADC-7/CR192 complex, the BATSI with the lowest Ki (0.45 nM) and greatest ΔTm (+9 °C), a trifluoromethyl substituent, interacts ...
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biochemical and structural analysis of inhibitors targeting the adc 7 Cephalosporinase of acinetobacter baumannii
Biochemistry, 2014Co-Authors: Rachel A Powers, Robert A Bonomo, Hollister C Swanson, Magdalena A Taracila, Nicholas W Florek, Chiara Romagnoli, Emilia Caselli, Fabio PratiAbstract:β-Lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived Cephalosporinases (ADCs). Currently, β-lactamase inhibitors are structurally similar to β-lactam substrates and are not effective inactivators of this class C Cephalosporinase. Herein, two boronic acid transition state inhibitors (BATSIs S02030 and SM23) that are chemically distinct from β-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal Cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 A resolution) and in complex with S02030 (2.0 A resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030...
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n152g s and t substitutions in cmy 2 β lactamase increase catalytic efficiency for cefoxitin and inactivation rates for tazobactam
Antimicrobial Agents and Chemotherapy, 2013Co-Authors: Marion J Skalweit, Magdalena A Taracila, Benjamin C Conklin, Rebecca A HuttonAbstract:Class C Cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in the Escherichia coli AmpC and P99 enzymes and have suggested that interactions between C-6′ or C-7′ substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 Cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A β-lactamase inhibitor with in vitro activity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC50) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C β-lactamase inhibitors are developed.
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exploring sequence requirements for c3 c4 carboxylate recognition in the pseudomonas aeruginosa Cephalosporinase insights into plasticity of the ampc β lactamase
Protein Science, 2011Co-Authors: Sarah M Drawz, Emilia Caselli, Magdalena A Taracila, Fabio Prati, Robert A BonomoAbstract:In Pseudomonas aeruginosa, the chromosomally encoded class C Cephalosporinase (AmpC β-lactamase) is often responsible for high-level resistance to β-lactam antibiotics. Despite years of study of these important β-lactamases, knowledge regarding how amino acid sequence dictates function of the AmpC Pseudomonas-derived Cephalosporinase (PDC) remains scarce. Insights into structure-function relationships are crucial to the design of both β-lactams and high-affinity inhibitors. In order to understand how PDC recognizes the C3/C4 carboxylate of β-lactams, we first examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor that possesses a side chain that mimics the thiazolidine/dihydrothiazine ring and the C3/C4 carboxylate characteristic of β-lactam substrates. We next tested the hypothesis generated by our model, i.e. that more than one amino acid residue is involved in recognition of the C3/C4 β-lactam carboxylate, and engineered alanine variants at three putative carboxylate binding amino acids. Antimicrobial susceptibility testing showed that the PDC-3 β-lactamase maintains a high level of activity despite the substitution of C3/C4 β-lactam carboxylate recognition residues. Enzyme kinetics were determined for a panel of nine penicillin and cephalosporin analog boronates synthesized as active site probes of the PDC-3 enzyme and the Arg349Ala variant. Our examination of the PDC-3 active site revealed that more than one residue could serve to interact with the C3/C4 carboxylate of the β-lactam. This functional versatility has implications for novel drug design, protein evolution, and resistance profile of this enzyme.