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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK013198-21
Application #
3224988
Study Section
Biochemistry Study Section (BIO)
Project Start
1978-04-01
Project End
1991-03-31
Budget Start
1989-07-01
Budget End
1991-03-31
Support Year
21
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
Hernandez, D; Phillips, A T (1994) Ser-143 is an essential active site residue in histidine ammonia-lyase of Pseudomonas putida. Biochem Biophys Res Commun 201:1433-8
King, R S; Sechrist, L L; Phillips, A T (1994) A revised map location for the histidine utilization genes in Pseudomonas putida. J Basic Microbiol 34:253-7
Hernandez, D; Phillips, A T; Zon, J (1994) 1-amino-2-imidazol-4'-ylethylphosphonic acid is a potent reversible inhibitor of Pseudomonas putida histidine ammonia-lyase. Biochem Mol Biol Int 32:189-94
Hernandez, D; Stroh, J G; Phillips, A T (1993) Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase. Arch Biochem Biophys 307:126-32
Hernandez, D; Phillips, A T (1993) Purification and characterization of Pseudomonas putida histidine ammonia-lyase expressed in Escherichia coli. Protein Expr Purif 4:473-8
Allison, S L; Phillips, A T (1990) Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Pseudomonas putida. J Bacteriol 172:5470-6
Consevage, M W; Phillips, A T (1990) Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida. J Bacteriol 172:2224-9
Hu, L; Allison, S L; Phillips, A T (1989) Identification of multiple repressor recognition sites in the hut system of Pseudomonas putida. J Bacteriol 171:4189-95
Hu, L; Phillips, A T (1988) Organization and multiple regulation of histidine utilization genes in Pseudomonas putida. J Bacteriol 170:4272-9
Hu, L; Mulfinger, L M; Phillips, A T (1987) Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida. J Bacteriol 169:4696-702