Beta-Lactamases

An In-Depth Look at the GES Family of Enzymes

Dr. Clyde Smith Jared MunozKwame Wiafe

Role and Identity

• Enzymes • Role in antibiotic breakdown

o Carbapenemso Penicillinso Cephamycins

• Gram negative and Gram positive bacteria

The GES Family

• GES-1 through GES-15o 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 Asn170o GES-5 is Gly170 to Ser170

  • Famous point group mutations

o  Sickle Cell Anemia Glu-6 to Val-6

o Tay-Sachs Diseaseo 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

Carbapenems1. 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.

ComparisonGES-2 GES-1

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

ComparisonGES-5 GES-1

Serine at position 166. Shown here are two conformations of Serine

Glycine in GES-1 at position 166.

Technique

• X-ray Crystallographyo 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 MADo SADo Molecular replacemento Multiple isomorphous replacemento Patterson function

• Molecular replacement- o 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 Detailedo 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 carbapenemso Caused by single point mutations

• Refinement shows lower occupancy of drugs in binding siteso Confirms the drug is being deacylated or released from

active site• Research is essential to creating new antibiotics

o Carbapenems are usually a last resorto More and more resistant bacteria

Sources1. 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, 982-9922. 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?