|Budget Amount *help
¥6,200,000 (Direct Cost : ¥6,200,000)
Fiscal Year 1993 : ¥2,400,000 (Direct Cost : ¥2,400,000)
Fiscal Year 1992 : ¥3,800,000 (Direct Cost : ¥3,800,000)
Some intermediates in the biosynthetic pathway generate an active pecies of oxygen via photochemical reactions upon illumination by visible light. For example, it is known that protoporphyrin IX, a intermediate in the biosynthesis of heme, is such a photosensitizer. Recently, Several light-sensitive mutant of E.coli have been isolated and characterized in my laboratory. The cells with mutantions in the visA (hemH) gene encoding ferrochelatase, the enzyme that catalyzes the final step in the biosynthesis of heme, are killed by illumination with visible light. The photoresistant mutants from the visA (hemH)-deleted strain have been isolated. Since on true revertant of visA (hemH) were expected, the revertats should be double mutants in the visA gene and in related genes that is involved in the heme biosynthetic pathway at a step before the reaction catayzed by ferrochelatase. In this research project, the studies on the genes involved in the biosynethsis of heme have been done in connection with light-responses of the cells.
(1) Using mutants isolated, the hemE and hemG gene of E.coli were cloned and sequenced.
(2) Two new genes involved in the biosynthesis of heme were identified. A new gene, designated hemK, composes a hemA-prfA-hemK operon at 27 min on the likage map. The hemK encodes 225 amino acids protein. Because the mutant cells accumulate protoporphyrinogen, the defect may locate in a gene functionally equvalent with the hemG gene.
(3) The cDNA clones of the hemA and hemH from barley and cucumber were isolated by a method of complementation with the visA-deleted or the hemA-deleted mutant of E.coli.
(4) A high expression system of the enzyme ferrochelatase was constructed and a large amount of the enzyme were prepared. The physical and biochemical natures of the purified enzyme were investigated. The crystallographic studies of this enzyme are under way.