Long term objectives To establish a detailed understanding of the enzymatic steps involved in the histidine biosynthetic pathway, leading ultimately to a knowledge of the organization of enzyme active sites in vivo. A focus of this investigation is to delineate the major mechanistic aspects of three catalytic steps in the pathway, and to relate this information to their protein structures. Medical Relevance. The knowledge gained from these studies will ultimately be critical for the design and selection of new antibacterial, antifungal or herbicidal agents targeted at primary events in a amino acid and purine metabolism. Because of the unique chemical features present in the metabolism under investigation, inhibitory agents directed at these steps would be operating by a novel mode of action. This feature is of particular importance in the development of antifungal that would be selective for a pathogen and nontoxic to a host.
Specific Aims 1. The mechanistic studies of the enzymatic amadori rearrangement catalyzed by 5'-ProFAR isomerase will be continued using hydrogen exchange experiments, substrate and product analogs as inhibitors. The appropriate preliminary investigations will be conducted to establish a method for arriving at a three dimensional structure of the enzyme using NMR and/or x-ray diffraction techniques. 2. The critical subunit interactions that mediate the glutamine amidotransferase properties of the imidazoleglycerol phosphate synthase will be discovered using a novel in vivo random mutagenesis screen. 3. A study of the sequence of reactions catalyzed by imidazoleglycerol phosphate synthase will be further examined. A major goal of this proposal will be the identification of the carbon-nitrogen ligation reactions which occur on this enzyme by using rapid-quench kinetics and mutant enzymes that are capable of partial turnover. 4. The E. coli phosphoribosyl-AMP cyclohydrolase encoded by the bifunctional hisIE gene will be purified for use in analog syntheses. A preparation of the substrate for this enzyme will be refined using a recombinant DNA system that encodes a mutant hisIE gene. The three dimensional structure of imidazoleglycerol phosphate dehydratase from Cryptococcus neoformans will be determined using x-ray crystallography. A tight-binding competitive inhibitor of the enzyme will be used in co-crystallizations to identify the active site and its relationship to the metal binding site.
| Klem, T J; Chen, Y; Davisson, V J (2001) Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase. J Bacteriol 183:989-96 |
| D'Ordine, R L; Klem, T J; Davisson, V J (1999) N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase: purification and characterization of a unique metalloenzyme. Biochemistry 38:1537-46 |
| Klem, T J; Davisson, V J (1993) Imidazole glycerol phosphate synthase: the glutamine amidotransferase in histidine biosynthesis. Biochemistry 32:5177-86 |
