This proposal concerns the regulation and enzymology of histidine utilization in Pseudomonas putida. This pathway serves a a source of one-carbon fragments for incorporation into purines and, in addition, a series of human genetic disorders have been described in which various enzymes of histidine utilization are affected. The pathway's initial enzyme, histidase (histidine ammonia-lyase), contains peptide-bound dehydroalanine as its coenzyme and one of the aims of the project is to elucidate the route whereby some amino acid in a precursor form of histidase becomes post-translationally modified to produce dehydroalanine. This will be achieved by a dual approach; one part is aimed at determining the amino acid sequence from data obtained on the DNA sequence of the histidase structural gene while a second phase examines the structure of histidase precursors generated by mutation in presumptive post-translational modification genes. The second enzyme in the histidine utilization pathway is urocanase (urocanate hydratase) and its structure is also being examined by sequencing of DNA corresponding to its structural gene. The amino acid sequence of urocanase will be extremely useful in understanding how NAD+, a required coenzyme for urocanase, functions in this reaction since it is likely that NAD works by a mechanism different from its usual role as an oxidation/reduction coenzyme. Mechanistic details of the urocanase reaction will be examined by transient state fluorescence analysis and 19F-NMR, using 2-fluorourocanic acid as a reporter substrate. The repressor protein controlling expression of the histidine utilizing genes will be isolated from an escherichia coli strain carrying an overproducing plasmid having the repressor gene cloned into it, with the intention of examining the ability of the repressor to recognize multiple inducers.
