In Pseudomonas putida the reactions of histidine degradation are carried out by five enzymes organized into apparently three operons, all under the control of a repressor whose synthesis is self-regulated. The first two enzymes, histidase and urocanase, respectively, along with the repressor protein, are being studied in this project because of their unusual coenzymatic functions or special properties. Histidase contains dehydroalanine which is essential for catalytic activity. The location and mode of attachment of this cofactor in histidase will be investigated by Edman degradation and mass spectrometry structural analysis of peptides which contain a labeled derivative of dehydroalanine. Mutants lacking dehydroalanine are to be used to identify the genes which are responsible for the incorporation of dehydroalanine (or its precursors) into histidase. Oligonucleotide- directed mutagenesis is also proposed to modify those attachment sites currently suspected in order to assess their role in the cross- bridged structure proposed. Because histidase can be produced from its P. putida gene cloned into Escherichia coli lacking this pathway, it is believed that the genes which specify dehydroalanine's attachment to histidase exist in E. coli as well as in P. putida. To establish this, the cloned histidase will be purified and its dehydroalanine structure compared to the P. putida enzyme. Mutants in post-translational modification genes will be sought in both species and the properties of their unmodified histidases studied to ascertain what has affected incorporation of dehydroalanine onto the protein. The promoter(s) and operator regions used for transcription of the various hut genes will be identified by DNA protection experiments and localized in the exact DNA sequence where known; the repressor protein's DNA sequence will be determined. Urocanase will be studied by transient state and rapid quench kinetic methods with several poor substrates in order to elucidate the role of NAD in the reaction mechanism. These studies should provide an improved understanding of the enzymology and regulation of this major degradative pathway, including biosynthesis and function of the novel dehydroalanine coenzyme in histidase and the unusual NAD addition complex mechanism believed to operate in the urocanase reaction.
