Date of Award
Doctor of Philosophy (PhD)
Alan J. Biel
Rhodobacter capsulatus uses the common tetrapyrrole pathway to synthesize heme, bacteriochlorophyll, siroheme and vitamin B-12. As part of our studies on the regulation of this pathway, the product of the hemC gene, porphobilinogen deaminase was purified approximately 200-fold. The molecular weight, isoelectric point, pH optimum and Km of the enzyme were all similar to other porphobilinogen deaminases. The stoichiometry of the enzyme was found not to be the expected four porphobilinogens used per one uroporphyrin formed. Rather a 9:1 ratio was typically seen. Further study of this atypical ratio uncovered evidence of possible in vivo regulation of porphobilinogen deaminase dependant on the cellular redox state. The protein was isolated from a two-dimensional gel and the sequence of the first twenty amino acids from the N-terminus was determined. This protein sequence matched a putative amino acid sequence 110 bp upstream of the R. capsulatus hemE gene. The putative hemC locus was subcloned from the plasmid pCAP154 which contains the R. capsulatus hemE gene with 1.2 Kb of upstream DNA. The sequence of the upstream DNA was determined by dideoxy sequencing of both strands. A single open reading frame of 951 bp was found which could code for a 317 amino acid protein of 34,082 Da. The putative protein has 49% identity with the E. coli porphobilinogen deaminase and 44% identity with the B. subtilis enzyme. When a plasmid carrying the R. capsulatus hemC was inserted into E. coli and R. capsulatus, a four-fold and ten-fold increase in porphobilinogen deaminase activity was seen respectively. The DNA sequence revealed that the hemC and hemE genes are divergently transcribed. A palindrome was identified between the two start sites, 47 bp upstream of hemE and 42 bp upstream of hemC. This palindrome, found upstream of many R. capsulatus genes, has been proposed to be a transcriptional regulatory site. The effects of oxygen on the transcription of hemC were studied using a hemC-cat fusion vector. Under the conditions tested, oxygen had no effect on the transcription of the R. capsulatus hemC gene.
Canada, Keith A., "Purification of Porphobilinogen Deaminase and the Sequencing and Expression of thehemC Gene of Rhodobacter Capsulatus." (1998). LSU Historical Dissertations and Theses. 6813.