| (delta)-Aminolevulinic acid (ALA), the first committed intermediate of the chlorophyll biosynthetic pathway, is synthesized from glutamate and is the limiting factor of the chlorophyll pathway. The enzymes from Chlamydomonas reinhardtii cells which are responsible for the formation of ALA from glutamate have been isolated and fractionated into three parts by serial affinity chromatography with a Blue Sepharose column and a heme-Sepharose column. The fractions are designated Blue-Sepharose bound (B), heme-Sepharose bound (H) and run-off (R). The enzymes require ATP, Mg('+2) and NADPH as cofactors for ALA formation.; Br(,s)-1, the brown mutant of Chlamydomonas, accumulates protoporphyrin (PROTO). The activity of its ALA synthesizing enzymes is less than one-eighth of that in wildtype cells. It is postulated that the accumulated PROTO in br(,s)-1 increases the heme level which in turn represses the ALA synthesizing enzymes. In the double mutant br(,s)-1 r-1, the specific activity of ALA synthesizing enzymes is more than three fold that from br(,s)-1 alone, but only about half that from wildtype. It is proposed that r-1 is a mutation of a regulatory gene and codes for a defective repressor. Consequently the ALA synthesizing enzymes of br(,s)-1 r-1 are partially de-repressed. Addition of the B fraction from wildtype to crude enzymes of br(,s)-1 and br(,s)-1 r-1 increases their activities, indicating that enzymes in the B fraction are limiting in these two strains. Furthermore, the enzyme(s) in the B fraction which converts glutamyl t-RNA to glutamate-1-semialdehyde (GSA) is sensitive to heme inhibition. Therefore the enzyme(s) converting glutamyl t-RNA to GSA is postulated to be regulated both by feedback inhibition and by repression.; The H fraction contains RNA which is the only component in the H fraction involved in the formation of ALA. RNA extracted from the H fraction as well as t-RNA('glu) from E. coli can convert glutamate to GSA when combined with the B fraction. It is postulated that glutamate is esterified to form glutamyl t-RNA by aminoacyl t-RNA synthetase in the presence of ATP and Mg('2+). Then a dehydrogenase converts glutamyl t-RNA to GSA using NADPH. Finally, catalyzed by an aminotransferase, GSA goes through intramolecular transamination to form ALA. |
