Histidine biosynthesis in plants and microorganisms occurs via an exclusive metabolic pathway that employs advanced metabolic precursors such as ATP and 5-phospho-D-ribosyl-1-pyrophosphate (PRPP) as carbon and nitrogen sources. Since this pathway is not present in mammals, the proposed research has important applications in the rational development of enzyme inhibitors with antibacterial, antifungal, and herbicidal applications. Abundant sequence alignment data indicate the similarity and evolutionary relationship of this enzyme in archaebacteria, eubacteria, and yeast. Thus, the specific aim of this proposal is to characterize N1-phosphoribosyl-AMP cyclohydrolase (HisI), an enzyme that catalyzes a critical and unique step in the biosynthesis of histidine: the hydrolysis of the purine ring of ATP. We will start by analyzing the specific requirements (e.g. metal ions), and steady state kinetic properties of monofunctional HisI from Methanococcus vannielii using a specific continuous UV-vis assay. The amino acid sequence alignment data and results of chemical modification experiments will be used to select specific amino acids as candidates for PCR mediated site specific mutagenesis in an effort to identify specific residues involved in catalysis. A desamino analog of N1-phosphoribosyl-AMP is proposed for use as potential inhibitor and mechanistic probe. Studies with the more complex, bifunctional E. coli HisIE enzyme will also be pursued once the details of the single function HisI are understood. The potential implication of these comparative studies into the evolution of enzyme catalysis is intriguing. Electrospray mass spectrometry, 18-O labeling studies and UV-vis will be employed in the investigation of the mechanism of this enzyme. A long range goal of this study is to evaluate HisI as a potential target for the design of specific inhibitors of histidine biosynthesis.
| D'Ordine, Robert L; Linger, Rebecca S; Thai, Carolyn J et al. (2012) Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase. Biochemistry 51:5791-803 |
| 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 |
