The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Intracellular proteins are degraded in all known cells. The mechanisms by which particular proteins are recognized for degradation, the pathways by which degradation occurs, and the nature and regulation of the enzymes involved are not well understood in any organism. In my lab, we use the well developed genetic systems of enteric bacteria along with biochemical and molecular biological methods to study these problems.

Roles of individual peptidases in peptide degradation. By isolating mutations that affect each of the major peptidase activities of Salmonella typhimurium, we have identified the genes and enzymes that act in the final steps of the degradation pathway to produce free amino acids from peptide intermediates. We would like to understand the specific role of each of these enzymes in the degradation pathway. Because some of these enzymes display a highly restricted substrate specificity, their function in the pathway is clear. Peptidases P and Q, for example, hydrolyze only N-terminal X-Pro bonds and are the only enzymes in the cell able to carry out this reaction. Other enzymes show a very broad substrate specificity hydrolyzing many types of peptide bonds. Peptidases A, B, and N are broad specificity aminopeptidases able to remove many different N-terminal amino acids from their substrates. We have found, however, that each of these enzymes has special capabilities not shared with any of the others. We would like to continue to explore the specificities of these enzymes, to understand the structural basis for these specificities, and to relate these properties to in vivo function.

Structure/function relationships in peptide hydrolyzing enzymes. Several of the peptidases we have found display unusual specificities. Peptidase E, for example, is absolutely specific for N-terminal Asp peptides and was the first enzyme identified with this property. It is also unusual in that its amino acid sequence does not place it in any of the known structural families of peptide hydrolyzing enzymes. We have recently found that Peptidase E is a new type of serine peptidase and have determined its 3-dimensional structure. We hope to use the structure to understand the basis of Peptidase E's unusual specificity. Other peptidases undergoing structural analysis include Peptidase T, an anaerobically regulated tripeptidase, and Oligopeptidase A. We plan to use both genetic selections and site-directed mutagnesis to obtain variant forms of these enzymes that illuminate the structural basis for their specificities.

Oligopeptidase A. Oligopeptidase A is metallopeptidase that hydrolyzes a variety of oligopeptides in vitro and catalyzes the N-terminal processing of a phage P22 protein (gp7) in vivo. Mutations (prlC) in the gene encoding this protein suppress the secretion defect conferred by certain signal sequence mutations. In addition, oligopeptidase A has been implicated in the degradation of signal peptides after they are released from the precursors of secreted proteins. The enzyme is related by amino acid sequence to large family of enzymes found in many bacteria and eukaryotes. opdA, the gene encoding oligopeptidase A, is part of the heat shock regulon. We are interested in understanding the role of oligopeptidase A in cell physiology. We are approaching this problem by attempting understand how oligopeptidase A recognizes gp7 and to use this information to find other cellular substrates. In processing gp7 oligopeptidase A seems indifferent to the amino acids present at the cleavage site recognizing instead structural features found elsewhere in the prosequence. We would like to understand the structural basis of this unusual recognition process and are attempting to determine the structures of both the enzyme and its substrate.