Enhancing resistance to cephalosporins in class C β-lactamases: Impact of Gly214Glu in CMY-2

Andrea Endimiani, Yohei Doi, Christopher R. Bethel, Magdalena Taracila, Jennifer M. Adams-Haduch, Alexandra O'Keefe, Andrea M. Hujer, David L. Paterson, Marion J. Skalweit, Malcolm G.P. Page, Sarah M. Drawz, Robert A. Bonomo

Research output: Contribution to journalArticle

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Abstract

The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Ω loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C β-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (kcat/Km) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k cat/Km against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone β-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both β-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is β-lactamase-catalyzed elimination of the C6 hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new β-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO32-.Molecular modeling and dynamics simulations suggest that the Ω loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis.When it is docked in the active site,we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems andmonobactams interact with CMY-2 and CMY-32 β-lactamases.

Original languageEnglish
Pages (from-to)1014-1023
Number of pages10
JournalBiochemistry
Volume49
Issue number5
DOIs
Publication statusPublished - 09-02-2010

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Cephalosporin Resistance
Cephalosporins
Monobactams
Enzymes
Carbapenems
Substitution reactions
Cefotaxime
meropenem
Amino Acid Substitution
doripenem
Catalytic Domain
Substrates
Aztreonam
Cephalothin
Sulbactam
Amino Acids
Electrospray ionization
Sulfones
Molecular modeling
Electrospray Ionization Mass Spectrometry

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Endimiani, A., Doi, Y., Bethel, C. R., Taracila, M., Adams-Haduch, J. M., O'Keefe, A., ... Bonomo, R. A. (2010). Enhancing resistance to cephalosporins in class C β-lactamases: Impact of Gly214Glu in CMY-2. Biochemistry, 49(5), 1014-1023. https://doi.org/10.1021/bi9015549
Endimiani, Andrea ; Doi, Yohei ; Bethel, Christopher R. ; Taracila, Magdalena ; Adams-Haduch, Jennifer M. ; O'Keefe, Alexandra ; Hujer, Andrea M. ; Paterson, David L. ; Skalweit, Marion J. ; Page, Malcolm G.P. ; Drawz, Sarah M. ; Bonomo, Robert A. / Enhancing resistance to cephalosporins in class C β-lactamases : Impact of Gly214Glu in CMY-2. In: Biochemistry. 2010 ; Vol. 49, No. 5. pp. 1014-1023.
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author = "Andrea Endimiani and Yohei Doi and Bethel, {Christopher R.} and Magdalena Taracila and Adams-Haduch, {Jennifer M.} and Alexandra O'Keefe and Hujer, {Andrea M.} and Paterson, {David L.} and Skalweit, {Marion J.} and Page, {Malcolm G.P.} and Drawz, {Sarah M.} and Bonomo, {Robert A.}",
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Endimiani, A, Doi, Y, Bethel, CR, Taracila, M, Adams-Haduch, JM, O'Keefe, A, Hujer, AM, Paterson, DL, Skalweit, MJ, Page, MGP, Drawz, SM & Bonomo, RA 2010, 'Enhancing resistance to cephalosporins in class C β-lactamases: Impact of Gly214Glu in CMY-2', Biochemistry, vol. 49, no. 5, pp. 1014-1023. https://doi.org/10.1021/bi9015549

Enhancing resistance to cephalosporins in class C β-lactamases : Impact of Gly214Glu in CMY-2. / Endimiani, Andrea; Doi, Yohei; Bethel, Christopher R.; Taracila, Magdalena; Adams-Haduch, Jennifer M.; O'Keefe, Alexandra; Hujer, Andrea M.; Paterson, David L.; Skalweit, Marion J.; Page, Malcolm G.P.; Drawz, Sarah M.; Bonomo, Robert A.

In: Biochemistry, Vol. 49, No. 5, 09.02.2010, p. 1014-1023.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Enhancing resistance to cephalosporins in class C β-lactamases

T2 - Impact of Gly214Glu in CMY-2

AU - Endimiani, Andrea

AU - Doi, Yohei

AU - Bethel, Christopher R.

AU - Taracila, Magdalena

AU - Adams-Haduch, Jennifer M.

AU - O'Keefe, Alexandra

AU - Hujer, Andrea M.

AU - Paterson, David L.

AU - Skalweit, Marion J.

AU - Page, Malcolm G.P.

AU - Drawz, Sarah M.

AU - Bonomo, Robert A.

PY - 2010/2/9

Y1 - 2010/2/9

N2 - The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Ω loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C β-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (kcat/Km) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k cat/Km against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone β-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both β-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is β-lactamase-catalyzed elimination of the C6 hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new β-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO32-.Molecular modeling and dynamics simulations suggest that the Ω loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis.When it is docked in the active site,we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems andmonobactams interact with CMY-2 and CMY-32 β-lactamases.

AB - The biochemical properties of CMY-32, a class C enzyme possessing a single-amino acid substitution in the Ω loop (Gly214Glu), were compared to those of the parent enzyme, CMY-2, a widespread class C β-lactamase. In parallel with our microbiological characterization, the Gly214Glu substitution in CMY-32 reduced catalytic efficiency (kcat/Km) by 50-70% against "good" substrates (i.e., cephalothin) while increasing k cat/Km against "poor" substrates (i.e., cefotaxime). Additionally, CMY-32 was more susceptible to inactivation by sulfone β-lactamase inhibitors (i.e., sulbactam and tazobactam) than CMY-2. Timed electrospray ionization mass spectrometry (ESI-MS) analysis of the reaction of CMY-2 and CMY-32 with different substrates and inhibitors suggested that both β-lactamases formed similar intermediates during catalysis and inactivation. We next showed that the carbapenems (imipenem, meropenem, and doripenem) form long-lived acyl-enzyme intermediates and present evidence that there is β-lactamase-catalyzed elimination of the C6 hydroxyethyl substituent. Furthermore, we discovered that the monobactam aztreonam and BAL29880, a new β-lactamase inhibitor of the monobactam class, inactivate CMY-2 and CMY-32 by forming an acyl-enzyme intermediate that undergoes elimination of SO32-.Molecular modeling and dynamics simulations suggest that the Ω loop is more constrained in CMY-32 than CMY-2. Our model also proposes that Gln120 adopts a novel conformation in the active site while new interactions form between Glu214 and Tyr221, thus explaining the increased level of cefotaxime hydrolysis.When it is docked in the active site,we observe that BAL29880 exploits contacts with highly conserved residues Lys67 and Asn152 in CMY-2 and CMY-32. These findings highlight (i) the impact of single-amino acid substitutions on protein evolution in clinically important AmpC enzymes and (ii) the novel insights into the mechanisms by which carbapenems andmonobactams interact with CMY-2 and CMY-32 β-lactamases.

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Endimiani A, Doi Y, Bethel CR, Taracila M, Adams-Haduch JM, O'Keefe A et al. Enhancing resistance to cephalosporins in class C β-lactamases: Impact of Gly214Glu in CMY-2. Biochemistry. 2010 Feb 9;49(5):1014-1023. https://doi.org/10.1021/bi9015549