Beta-Lactamases

1. 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? • 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, 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 Crystallography60: 2126-2132 Part 12 Sp. Iss. 1 DEC 2004  5. General Reference: http://www.acsmedchem.org/module/betalactam.html

17. Questions?