Improving Antibiotics

Biological macromolecules are amazingly complex and diverse. Understanding the relationship between the structure and function of proteins is key to understanding biological processes. Our laboratory has been using X-ray diffraction to determine the atomic-level, three-dimensional structures of D-alanyl-D-alanine peptidases. These enzymes are involved in the final stages of bacterial cell-wall biosynthesis where new cell wall strands are cross-linked to form the intact cell wall. The reaction catalyzed by DD-peptidases is inhibited by ß-lactam antibiotics (penicillins and cephalosporins). ß-lactams are effective drug molecules because they mimic the natural substrate for the enzymes, the cell wall D-alanyl-D-alanine components. When you inhibit the DD-peptidases, growing bacteria are unable to cross-link their cell wall, and the bacteria lyse and die.

By determining the structure of a DD-peptidase and studying complexes formed between the enzyme and different penicillins and cephalosporins, we are gaining insight into the mechanism of action of this important class of drug molecules. This understanding can, in turn, be used to guide the design of new antibacterial compounds. This is significant because bacteria are becoming increasingly resistant to ß-lactams primarily through the production of enzymes called ß-lactamases that are able to break down penicillins and cephalosporins. With structural information in hand, medicinal chemists and crystallographers can work together to develop new drugs that will bind tightly to the DD-peptidases and evade the ß-lactamases.

To determine the structure of a protein, one must first purify the protein to near homogeneity and crystallize it. There is no recipe book for protein crystallization; it takes great care and patience and some luck. Once you have good crystals, you expose them to X rays and collect the scattered radiation data. Those data contain the information that allows you to go back and build a model of the protein. Using interactive computer graphics, you display the experimental data and fit the data with the amino acid residues that make up the protein. We have carried out these experiments for the DD-peptidase from the bacterium Streptomyces R61 and have determined the structure of this enzyme to 1.6Å resolution (very close to the size of individual atoms). We have studied the binding of drug molecules to the active site of the enzyme and are beginning to understand how this enzyme works.

Two undergraduate students in my laboratory, Carole Capen and Steve Condron, and I are working on the DD-peptidase from another bacterial source. This enzyme shows a different sensitivity to various ß-lactams. Carole and Steve are working on purifying milligram quantities of enzyme and on the crystallization of these samples. We hope that when we solve the structure of this DD-peptidase we will gain further insight into subtle variations in the active sites of the enzymes that result in altered specificity.