1. 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

2. Role and Identity Enzymes Role in antibiotic breakdown Carbapenems
Penicillins
Cephamycins
Gram negative and Gram positive bacteria

3. 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

4. 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

5. Beta-lactam ring General core structure of penicillins
General core structure of cephalosporins

6. Carbapenems
1. Imipenem
2. Ertapenem
3. Meropenem

7. 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.

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

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

10. 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

11. 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.

12. 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

13. Active Site with Ertapenem
GES-2 with Ertapenem

14. Active Site with Ertapenem 2
GES-5 and Ertapenem

15. 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

16. 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,
2. Source of Images for Imipenem, Meropenem, Ertapenem and General Strucutre of Beta-lactams: 
   
   
   
   
   
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: Part 12 Sp. Iss. 1 DEC 2004 
5. General Reference:

17. Questions?