Tetrapyrrole synthesis in Bacillus subtilis
Abstract: This thesis describes tetrapyrrole synthesis in the aerobic Gram positive bacterium Bacillus sub-tilis. Tetrapyrroles such as heme of a-, b-, c-, d- and o-type can be synthesised in this organism. In addition, there is strong evidence for the presence of the tetrapyrrole siroheme in B. subtilis. Heme b (protoheme IX) and siroheme are synthesised in ten and nine enzymatic steps, respectively, from eight molecules of glutamate. The initial steps from glutamyl-tRNA to uroporphyrinogen III are common to these tetrapyrroles and catalysed by the hemA, hemL, hemB, hemC and hemD gene products, respectively. These genes are found in the hemAXCDBL operon. Another operon, hemEHY, contains genes for “late” steps of heme synthesis; from uroporphyrinogen III to heme b. The first part of the thesis concerns the cloning and sequencing of genes encoding tetrapyrrole biosynthetic enzymes in B. subtilis. Gene products were identified and the orga-nisation of the genes involved in the heme biosynthetic pathway was analysed. In the final part, two enzymes were studied in more detail; ferrochelatase and the HemY protein. Ferrochelatase is encoded by hemH and catalyses the insertion of ferrous iron into protoporphyrin IX generating heme b. B. subtilis ferrochelatase differs from other ferrochelatases in being a water soluble enzyme and in that it can incorporate Cu2+ into protoporphyrin IX. It appears to be a “slow” enzyme with a turnover number of 18-29 per minute but this rate exceeds the rate of heme synthesis in exponentially growing B. subtilis which was estimated to be 0.2 mol heme formed per minute and mol of enzyme. B. subtilis hemY encodes a peripheral membrane-bound enzyme in B. subtilis and also when expressed in E. coli. HemY has protoporphyrinogen IX oxidase activity; i.e. it catalyses the penultimate step in heme biosynthesis.
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