An In-Depth Look at the GES Family of Enzymes 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 Carbapenems Penicillins Cephamycins Gram negative and Gram positive bacteria

The GES Family GES-1 through GES-15 GES-2 through GES-15 are all point mutants of GES-1 GES-2 and GES-5 are single AA mutations at residue 170. GES-2 is Gly170 to Asn170 GES-5 is Gly170 to Ser170   Famous point group mutations  Sickle Cell Anemia Glu-6 to Val-6 Tay-Sachs Disease Cystic Fibrosis

How do they work? Reasonable threat... Bind and break down beta-lactam rings Indirectly destroys bacterial cell wall Deacylation renders drug inactive Reasonable threat... Antibiotics are our first line of defense against bacterial diseases. B-lactamases are shortening the list of effective antibiotics Another issue:  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 Beta-lactam ring. This action prevents binding of antibiotic to PBP.

Comparison GES-2 GES-1 Asparagine at position 166 in GES-2. Glycine in GES-1 at position 166

Comparison GES-5 GES-1 Serine at position 166. Shown here are two conformations of Serine Glycine in GES-1 at position 166.

Technique X-ray Crystallography X-rays diffract Bragg's Law nλ = 2d sinΘ Creates diffraction pattern Governed by components of crystal Resulting Intensities Construction of electron density maps

The Phase Problem Loss of information concerning a phase Methods MAD SAD Molecular replacement Multiple isomorphous replacement Patterson function Molecular replacement-  First a similar model is needed  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 Detailed 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 Caused by single point mutations Refinement shows lower occupancy of drugs in binding sites Confirms the drug is being deacylated or released from active site Research is essential to creating new antibiotics Carbapenems are usually a last resort 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 extended-spectrum 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

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