Bauerle, Ronald H.
Primary Appointment
Professor Emeritus, Biology
Contact Information
Telephone: 434-982-5477
Email: rhb7g@virginia.edu
Research Interests
Structure, Function and Evolution of Allosteric Enzymes
Research Description
The main research interest of my laboratory is the molecular basis for structure, function, regulation and evolution in allosteric enzymes. We are presently studying three different enzyme systems from the aromatic and lipopolysaccharide biosynthetic pathways in enteric bacteria.
Regulation of carbon flow in the various branches of aromatic biosynthesis is achieved by a series of feedback loops, modulated by the intracellular pool levels of the three aromatic amino acid end products, namely L-phenylalanine, L-tryptophan and L-tyrosine. This feedback control is mediated by the allosteric properties of a number of key pathway enzymes whose activities are specifically inhibited by the binding of one of the aromatic amino acids. Among these regulatory enzymes is DAHP synthase, which is responsible for the first committed step of the common aromatic pathway, and the anthranilate synthase-phosphoribosyl transferase complex (AS-PRT), a multifunctional enzyme which catalyzes the first two reactions of the tryptophan pathway. A third enzyme of interest to us, KDOP synthase, catalyzes an essential step in the biosynthesis of the lipopolysaccharide endotoxin located on the outer membrane of the bacterial cell.
These three enzymes have unique features that make them attractive model systems for structure-function analysis. DAHP synthase exists in the cell as three isozymes. Although catalyzing the same reaction, each isozyme is specifically feedback regulated by one of the three aromatic amino acids. The AS-PRT is a tetrameric complex made up of two dissimilar subunits, both of which have multiple functional domains. All of the enzymatic activities of the complex are feedback-regulated by tryptophan, resulting from the conformational effects of its binding to a regulatory domain in one of the two subunits. The KDOP synthase has striking similarities in mechanism and structure with the DAHP synthases, suggesting a common ancestry.
Our work entails parallel analysis of the molecular architecture of the AS-PRT complex, the three DAHP synthase isozymes and the KDOP synthase. Our aim is to establish the structural basis for the functional and regulatory properties unique to each and to learn whether similar properties, such as the binding of identical ligands, have a common structural basis and possibly a common evolutionary origin. In this work we apply a variety of genetic, biochemical and biotechnological approaches, analyzing both the genes and the proteins. These include in vitro mutagenesis, protein engineering, DNA and peptide sequencing, protein purification and enzymatic characterization using a variety of kinetic, spectroscopic, electrophoretic and chromatographic methodologies. In addition, three dimensional structural analysis of the enzymes by x-ray crystallography is pursued in collaboration with the laboratory of Robert Kretsinger.