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Beta-Lactamases
An In-Depth Look at the GES Family
of Enzymes
Dr. Clyde Smith
Jared Munoz
Kwame Wiafe
Role and Identity
• Enzymes
• Role in antibiotic breakdown
o Carbapenems
o Penicillins
o Cephamycins
• Gram negative and Gram positive bacteria
The GES Family
• GES-1 through GES-15
o GES-2 through GES-15 are all point mutants of GES-1
• GES-2 and GES-5 are single AA mutations at residue 170.
o GES-2 is Gly170 to Asn170
o GES-5 is Gly170 to Ser170
• Famous point group mutations
o Sickle Cell Anemia
 Glu-6 to Val-6
o Tay-Sachs Disease
o Cystic Fibrosis
How do they work?
• Bind and break down beta-lactam rings
o Indirectly destroys bacterial cell wall
• Deacylation renders drug inactive
Reasonable threat...
• Antibiotics are our first line of defense against bacterial
diseases.
o B-lactamases are shortening the list of effective antibiotics
• Another issue:
o genetic information that codes for B-lactamases can be
transferred amongst bacteria
Beta-lactam ring
General core structure of
penicillins
General core structure
of cephalosporins
Carbapenems
1. Imipenem
2. Ertapenem
3. Meropenem
Binding to Penicilling Binding Protein
Penicillin binding Protein is
also known as Cell Wall
Transamidase.
Covalent bond between PBP
and beta-lactam ring on
antibiotic would render the
protein uselss.
Beta-lactamases take care of
this by breaknig the Betalactam ring. This action
prevents binding of antibiotic
to PBP.
Comparison
GES-2
Asparagine at position 166 in GES-2.
GES-1
Glycine in GES-1 at position 166
Comparison
GES-5
Serine at position 166. Shown here are two
conformations of Serine
GES-1
Glycine in GES-1 at position 166.
Technique
• X-ray Crystallography
o X-rays diffract
 Bragg's Law
 nλ = 2d sinΘ
o Creates diffraction pattern
 Governed by components of crystal
 Resulting Intensities
o Construction of electron density maps
The Phase Problem
• Loss of information concerning a phase
• Methods
o MAD
o SAD
o Molecular replacement
o Multiple isomorphous replacement
o Patterson function
• Molecular replacemento First a similar model is needed
o Since the structure of GES-1 is known, GES-2 and GES-5
crystal structures were solved using GES-1.
Modeling/Refinement
• Modeling program, Coot, was used to examine crystal
structures of GES-1, GES-2 and GES-5
• Refinment program allows refinement based on generated
density maps
• Protein model is loaded
• Phenix Refinement
o Detailed
o Generates a new map based on current structure
Active Site with Ertapenem
GES-2 with Ertapenem
Active Site with Ertapenem 2
GES-5 and Ertapenem
Conclusion
• GES-2 and GES-5 have increased binding and activity for
carbapenems
o Caused by single point mutations
• Refinement shows lower occupancy of drugs in binding sites
o Confirms the drug is being deacylated or released from
active site
• Research is essential to creating new antibiotics
o Carbapenems are usually a last resort
o More and more resistant bacteria
Sources
1. Structure of GES-1 at atomic resolution: insights into the
evolution of carbapenemase activity in the class A extendedspectrum B-lactamases. Smith, Clyde A., Caccamo, Marissa,
Kantardjieff, Katherine A. et al. Acta Cryst. (2007). D63, 982-992
2. Source of Images for Imipenem, Meropenem, Ertapenem and
General Strucutre of Beta-lactams:
http://en.wikipedia.org/wiki/File:Imipenem.svg
http://en.wikipedia.org/wiki/File:Ertapenem.svg
http://en.wikipedia.org/wiki/File:Meropenem.svg
http://en.wikipedia.org/wiki/File:Beta-lactam_antibiotics_example_1.svg
http://commons.wikimedia.org/wiki/File:Penicillin_inhibition.svg
3. The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.
4. "Coot: model-building tools for molecular graphics" Emsley P, Cowtan K Acta
Crystallographica Section D-Biological Crystallography 60: 2126-2132 Part 12 Sp.
Iss. 1 DEC 2004
5. General Reference: http://www.acsmedchem.org/module/betalactam.html
Questions?