Oxygen-Mediated Regulation of Tetrapyrrole Synthesis in Rhodobacter Capsulatus

[304999] Biel本研究的最终目的是阐明氧调控荚膜红杆菌中四吡咯生物合成的机制。最近的证据表明，两个氨基乙酰丙酸酯分子的缩合形成第一个吡啶中间体，卟啉原，是由氧调节的。这一步骤是由血红蛋白b基因的产物——促胆红素原合成酶催化的。本提案中描述的研究旨在确定氧气是否调节编码卟啉胆碱原合成酶的hemB基因的转录，或该酶的活性，或两者兼而有之。hemB基因的转录调控将通过测量在高氧和低氧培养中hemB mRNA的产生和降解来研究。氧介导的卟啉原合成酶活性的调节将通过在氧张力升高期间跟踪原卟啉形成的动力学来测量。如果氧调节卟胆色素原合成酶的活性，原卟啉的积累应在培养物充氧后立即停止。针对卟啉胆色素原合成酶的抗体将用于确定氧气是否会导致酶的降解增加。在光合细菌荚膜红杆菌中，单支途径用于制造血红素和细菌叶绿素。氧气极大地减少了这一途径的表达。氧气调节这一途径的步骤已经确定，本提案中描述的研究旨在确定氧气是否调节卟胆色素原合成酶的合成，酶的活性，或两者兼而有之。＊＊＊

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. ***