Hughes, Lee E., Pyrimidine biosynthesis in the genus Streptomyces: Characterization of aspartate transcarbamoylase and its interaction with other pyrimidine enzymes. Doctor of Philosophy (Microbiology), May, 1998, 188 pp., 16 tables, 43 illustrations, references, 181 titles.

Aspartate transcarbamoylase (ATCase) of Streptomyces was characterized and its interaction with other pyrimidine enzymes explored. ATCase and dihydroorotase (DHOase) of S. griseus were assayed and purified using conventional methods and HPLC. The two activities were found to co-purify, suggesting both are found in a complex. The S. griseus holoenzyme has an estimated molecular mass of 480 kDa. Examination by SDS-PAGE revealed that the holoenzyme is composed of two types of subunits. Western blot analysis using antibody to E. coli PyrB (ATCase catalytic subunit) showed a cross reaction with the 38 kDa subunit from the S. griseus ATCase/DHOase complex. Only two other bacterial ATCases, from Deinococcus and Thermus, have been reported to contain both ATCase and DHOase catalytically-active subunits. The similarity of these ATCases to that of S. griseus is surprising considering the wide divergence between these organisms and streptomycetes.

The S. griseus ATCase showed typical Michaelis-Menten kinetics for velocity versus substrate plots for both aspartate and carbamoylphosphate. These kinetics and the observed molecular mass place Streptomyces ATCase in the Class A bacterial ATCases. Like other Class A enzymes, S. griseus ATCase was inhibited by ATP, CTP, and UTP.

Interactions between the biosynthetic and salvage pathways were also found. A 5-fluorouracil resistant mutant of S. griseus was selected which lacked uracil phosphoribosyltransferase (Upp) activity. This strain was found to be derepressed 8-10 fold for ATCase and DHOase. Furthermore, ATCase in this strain was more sensitive to effector molecules than was the wild-type enzyme. Considering the positive selection method used, there is likely to be a mutation in only one gene, upp. Therefore, the observed loss of Upp activity, derepression of the pathway, and increased effector sensitivity are linked. This would be possible only if the same polypeptide were involved in each.

Genetic studies using PCR showed that the Streptomyces pyrimidine operon is organized similarly to that of Mycobacterium.

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