STRUCTURE, FUNCTION AND GENETIC REGULATION OF AMIDOPHOSPHORIBOSYL TRANSFERASE
Abstract
The regulation of Escherichia coli purF was studied in order to gain a better understanding of pur regulon gene expression. E. coli purF has been determined to be the distal gene of a polycistronic operon. The first gene of the purF operon encodes a 17.9 kDa hydrophobic protein of unknown function. mRNA hybidization studies establish purF is regulated at the transcriptional level. The purF promoter and control region were identified using S1 mapping, in vitro transcription and a deletion analysis. Sequences required for purF regulation have been localized to a region surrounding the transcription start site. The regulation of E. coli purF is complex, most likely including pur specific repressor binding and stringent control. To gain information on amidophosphoribosyltransferase structure-function relationships, Bacillus subtilis purF was cloned and sequenced. A primary translation product of 476 amino acids was deduced from the DNA sequence. Analysis of the deduced amino acid sequence identified an 11 amino acid N-terminal extension that is processed off to yield the mature enzyme and the ligand binding site for a 4Fe-4S cluster. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphotransferase. Even though amidophosphoribosyltransferase from E. coli does not contain an FeS cluster or undergo N-terminal processing, the plasmid encoded enzyme, produced in E. coli, was found to be identical to the enzyme isolated from B. subtilis. To investigate the role of the FeS cluster in B. subtilis amidophosphoribosyltransferase, five mutations in and around the 4Fe-4S binding site were constructed using site-directed mutagenesis and integrated into the B. subtilis chromosome. Analysis of the mutants confirmed a previous assignment of the ligand binding site and revealed that correct binding of the FeS cluster is required for enzyme activity and N-terminal processing. However, the FeS cluster is not involved in catalysis or allosteric regulation. Pulse-chase experiments indicate that N-terminal processing is a slow, post-translational event that most likely involves cellular factors.
Degree
Ph.D.
Subject Area
Molecular biology
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