9304999 Biel The ultimate objective of this research is to elucidate the mechanism by which oxygen regulates tetrapyrrole biosynthesis in Rhodobacter capsulatus. Recent evidence has suggested that the condensation of two aminolevulinate molecules to form the first pyrrolic intermediate, porphobilinogen, is regulated by oxygen. This step is catalyzed by the enzyme prophobilinogen synthase, the product of the hemB gene. The research described in this proposal seeks to determine whether oxygen regulates transcription of the hemB gene, which encodes porphobilinogen synthase, or the activity of this enzyme, or both. Transcriptional regulation of the hemB gene will be investigated by measuring hemB mRNA production and degradation in cultures grown under high and low oxygen. Oxygen- mediated regulation of porphobilinogen synthase activity will be measured by following the kinetics of protoporphyrin formation during a shift-up in oxygen tension. If oxygen regulates porphobilinogen synthase activity, protoporphyrin accumulation should stop immediately upon oxygenation of the culture. Antibodies against porphobilinogen synthase will be used to determine if oxygen causes increased degradation of the enzyme. %%% In the photosynthetic bacterium Rhodobacter capsulatus, a single branched pathway is used to make heme and bacteriochlorophyll. Oxygen greatly reduced the expression of this pathway. The step at which oxygen regulates this pathway has been located, and the research described in this proposal seeks to determine whether oxygen regulates the synthesis of the enzyme porphobilinogen synthase, the activity of the enzyme, or both. ***
