BETA LACTAMASE MUTATIONS IN ANTIBIOTIC RESISTANCE

Summary

Principal Investigator: Timothy Palzkill
Abstract: Role of p-lactamase Mutations in Antibiotic Resistance Bacterial resistance to antimicrobial agents has increased in recent years and now represents a significant threat to successful antibiotic therapy. One example of this phenomenon is the development of resistance to p-lactam antibiotics, p-lactam antibiotics, such as the penicillins and cephalosporins, are among the most frequently used antimicrobialagents. The most common mechanism of resistance to P-lactam antibiotics is the production of p-lactamases, which cleave the antibiotic, rendering it harmless to bacteria. Based on primary sequence homology, p-lactamases have been grouped into four classes. Classes A, C and D arc active-siteserinc enzymes that catalyze, via a serine-bound acyl-enzyme intermediate,the hydrolysis of the p-lactam antibiotic. Class B enzymes require zinc for activity and catalysis does not proceed via a covalent intermediate.Because of the diverse range of substrate specificities of these enzymes, virtually all p-lactam antibioticsaresusceptible to hydrolysis. Clearly, the design of new antibiotics that escape hydrolysis by the growing collectionof \\- lactamase activitieswill be a challenge. It will be necessary to understand the catalytic mechanism and basis for substrate specificity of each class of p-lactamase. The goal of this work is understand how the amino acid sequence determines the structure, catalysis, and substrate specificityof the IMP-1 p-lactamase of class B and the P99 class C p-lactamasc. This will be achieved by randomizingamino acid positions in theactive-site pocket of each en/yme to sample all possible aminoacid substitutions. All of the random substitutions will then be screened to identify those substitutionsthat alter the substrate specificity of the enzyme. Enzymes containing substitutions that alter substrate specificitywill be purified and characterized biochemically. The sets of random substitutionswill also be screened using phage display methodology to identify residues critical for catalysis. A further goal of this proposal is to use the detailed knowledge of the interface between p- lactamase inhibitoryprotein (BLIP) and p-lactamasc, in combinationwith random mutagenesis and phage display, to create derivatives of BLIP that bind and inhibit P-lactamases and penicillin binding proteins. The new BLIP derivatives will be characterized biochemically and structurally. The informationgained from these studies will be useful for the rational design of new antibioticsand inhibitors.
Funding Period: ----------------1992 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. ncbi Thermodynamic investigation of the role of contact residues of beta-lactamase-inhibitory protein for binding to TEM-1 beta-lactamase
    Jihong Wang
    Department of Chemistry, University of Houston, Houston, Texas 77204 5003, USA
    J Biol Chem 282:17676-84. 2007
  2. pmc BLIP-II is a highly potent inhibitor of Klebsiella pneumoniae carbapenemase (KPC-2)
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology and Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA
    Antimicrob Agents Chemother 57:3398-401. 2013
  3. pmc Metallo-β-lactamase structure and function
    Timothy Palzkill
    Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA
    Ann N Y Acad Sci 1277:91-104. 2013
  4. pmc Deep sequencing of systematic combinatorial libraries reveals β-lactamase sequence constraints at high resolution
    Zhifeng Deng
    Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    J Mol Biol 424:150-67. 2012
  5. pmc Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site
    Lori B Horton
    Departments of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
    Antimicrob Agents Chemother 56:5667-77. 2012
  6. pmc Identification of the β-lactamase inhibitor protein-II (BLIP-II) interface residues essential for binding affinity and specificity for class A β-lactamases
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
    J Biol Chem 288:17156-66. 2013
  7. pmc A fitness cost associated with the antibiotic resistance enzyme SME-1 beta-lactamase
    David C Marciano
    Department of Molecular Virology and Microbiology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA
    Genetics 176:2381-92. 2007
  8. pmc Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 beta-lactamase
    David C Marciano
    Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
    J Mol Biol 384:151-64. 2008
  9. pmc Structural and biochemical evidence that a TEM-1 beta-lactamase N170G active site mutant acts via substrate-assisted catalysis
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
    J Biol Chem 284:33703-12. 2009
  10. pmc Structural insight into the kinetics and DeltaCp of interactions between TEM-1 beta-lactamase and beta-lactamase inhibitory protein (BLIP)
    Jihong Wang
    Department of Chemistry, University of Houston, Houston, Texas 77204 5003, USA
    J Biol Chem 284:595-609. 2009

Detail Information

Publications19

  1. ncbi Thermodynamic investigation of the role of contact residues of beta-lactamase-inhibitory protein for binding to TEM-1 beta-lactamase
    Jihong Wang
    Department of Chemistry, University of Houston, Houston, Texas 77204 5003, USA
    J Biol Chem 282:17676-84. 2007
    ..Finally, the affinity enhancing effect of the contact residue mutant Y50A may be due to energetic coupling interactions between Asp(49) and His(41)...
  2. pmc BLIP-II is a highly potent inhibitor of Klebsiella pneumoniae carbapenemase (KPC-2)
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology and Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA
    Antimicrob Agents Chemother 57:3398-401. 2013
    ..Determination of association and dissociation rate constants for binding between BLIP-II and KPC-2 reveals a very tight interaction with a calculated (koff/kon) equilibrium dissociation constant of 76 fM (76 × 10(-15) M)...
  3. pmc Metallo-β-lactamase structure and function
    Timothy Palzkill
    Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA
    Ann N Y Acad Sci 1277:91-104. 2013
    ..This review summarizes the numerous studies that have yielded insights into the structure, function, and mechanism of action of these enzymes...
  4. pmc Deep sequencing of systematic combinatorial libraries reveals β-lactamase sequence constraints at high resolution
    Zhifeng Deng
    Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    J Mol Biol 424:150-67. 2012
    ..An analysis of the TEM-1 and other class A structures suggests that residues that vary in the alignment may nevertheless make unique, but important, interactions within individual enzymes...
  5. pmc Mutagenesis of zinc ligand residue Cys221 reveals plasticity in the IMP-1 metallo-β-lactamase active site
    Lori B Horton
    Departments of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA
    Antimicrob Agents Chemother 56:5667-77. 2012
    ..Finally, substitutions at residue 121, which is the IMP-1 equivalent of the subclass B3 zinc-chelating position, failed to rescue C221G function, suggesting the coordination schemes of subclasses B1 and B3 are not interchangeable...
  6. pmc Identification of the β-lactamase inhibitor protein-II (BLIP-II) interface residues essential for binding affinity and specificity for class A β-lactamases
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
    J Biol Chem 288:17156-66. 2013
    ..e., a substitution that reduces affinity for one β-lactamase usually reduces affinity for all β-lactamases tested...
  7. pmc A fitness cost associated with the antibiotic resistance enzyme SME-1 beta-lactamase
    David C Marciano
    Department of Molecular Virology and Microbiology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA
    Genetics 176:2381-92. 2007
    ..Taken together, these results suggest that fitness costs associated with some beta-lactamases may limit their dissemination...
  8. pmc Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 beta-lactamase
    David C Marciano
    Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
    J Mol Biol 384:151-64. 2008
    ..Collectively, the data suggest that L201P promotes tolerance of some deleterious TEM-1 mutations by enhancing the protein stability of these mutants...
  9. pmc Structural and biochemical evidence that a TEM-1 beta-lactamase N170G active site mutant acts via substrate-assisted catalysis
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
    J Biol Chem 284:33703-12. 2009
    ....
  10. pmc Structural insight into the kinetics and DeltaCp of interactions between TEM-1 beta-lactamase and beta-lactamase inhibitory protein (BLIP)
    Jihong Wang
    Department of Chemistry, University of Houston, Houston, Texas 77204 5003, USA
    J Biol Chem 284:595-609. 2009
    ..It appears that the increase of interface-trapped water molecules is inversely correlated with negative binding heat capacity changes...
  11. pmc Fine mapping of the sequence requirements for binding of beta-lactamase inhibitory protein (BLIP) to TEM-1 beta-lactamase using a genetic screen for BLIP function
    Ji Yuan
    Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    J Mol Biol 389:401-12. 2009
    ..The results indicate the BLIP surface is tolerant of substitutions in that many contact positions can be substituted with other amino acid types and retain wild-type levels of function...
  12. pmc Use of periplasmic target protein capture for phage display engineering of tight-binding protein-protein interactions
    Bartlomiej G Fryszczyn
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    Protein Eng Des Sel 24:819-28. 2011
    ....
  13. pmc Analysis of the functional contributions of Asn233 in metallo-β-lactamase IMP-1
    Nicholas G Brown
    Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA
    Antimicrob Agents Chemother 55:5696-702. 2011
    ....
  14. pmc Analysis of the binding forces driving the tight interactions between beta-lactamase inhibitory protein-II (BLIP-II) and class A beta-lactamases
    Nicholas G Brown
    Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030, USA
    J Biol Chem 286:32723-35. 2011
    ..The structural rigidity of BLIP-II minimizes the loss of the entropy upon complex formation and, as indicated by thermodynamics experiments, may be a key determinant of the observed potent inhibition of β-lactamases...
  15. pmc Co-crystal structures of PKG Iβ (92-227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding
    Jeong Joo Kim
    Department of Pharmacology, Baylor College of Medicine, Houston, Texas, United States of America
    PLoS ONE 6:e18413. 2011
    ....
  16. pmc Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM-1 beta-lactamase
    David C Marciano
    Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA
    Protein Sci 18:2080-9. 2009
    ..These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A beta-lactamases...
  17. pmc Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM β-lactamases
    Nicholas G Brown
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    J Mol Biol 404:832-46. 2010
    ..The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function...
  18. pmc Identification and characterization of beta-lactamase inhibitor protein-II (BLIP-II) interactions with beta-lactamases using phage display
    N G Brown
    Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    Protein Eng Des Sel 23:469-78. 2010
    ..The phage system, as well as enzyme inhibition assays with purified proteins, revealed that BLIP-II is a more potent inhibitor than BLIP for several class A beta-lactamases with K(i) values in the low picomolar range...
  19. pmc Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase
    Ji Yuan
    Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
    J Mol Biol 406:730-44. 2011
    ....