Biosynthesis of Tetrapyrrole Pigment Precursors

Abstract

The aminotransferase that catalyzes the formation of 6-aminolevulinic acid from glutamate-1-semialdehyde or from glutamate in a reconstituted enzyme system was isolated and partially purified from Chlorella vulgaris. The apparent molecular weight of the aminotransferase was determined by Sephadex G-100 and Ultrogel AcA 54 gel filtration to be 60,000 ± 5,000. Catalytic activity of the aminotransferase required pyrixodal phosphate (PALP). The cofactor could not be removed by gel filtration after exposure of the enzyme to PALP. Aminotransferase was inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The concentration of gabaculine required for half maximal inhibition was about 0.05 micromolar. Aminotransferase activity could be regained upon the removal of gabaculine by gel filtration and supplementing the assay medium with PALP. Neither the inhibitory action of gabaculine nor its reversibility was affected by preincubation of the enzyme with the keto acids levulinate and &-aminolevulinic acid.the reaction to inhibitors that are considered to be pyridoxal antagonists, such as gabaculine and aminooxyacetate (5, 10, 13, 21). However, previous attempts to demonstrate directly a dependence of enzyme activity on PALP were unsuccessful (6,20). The apparent lack of dependence of the aminotransferase on PALP was explained as resulting from the existence of a tightly bound pyridoxal or pyridoxamine phosphate that is not removed from the enzyme by the various purification procedures employed during the preparation of the enzyme extract (5).We now report the direct demonstration of the dependence of aminotransferase activity in vitro on added PALP and the positive effect ofPALP on the recovery ofaminotransferase activity following preincubation of the enzyme with gabaculine.

Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit

“…molecules from various sources (1,7,17,18,32), and the cognate tRNA for the GTR being studied is often not available.…”

Intermolecular Nitrogen Transfer in the Enzymic Conversion of Glutamate to [delta]-Aminolevulinic Acid by Extracts of Chlorella vulgaris

“…Of the two remaining proposed reaction models, both of which have enzyme-bound DAVA as the intermediate (Fig. 2, models 2 and 3), model 2 is preferred on the basis of the high effectiveness of the mechanismbased suicide inhibitor, gabaculine, which inhibits the GSA aminotransferase by reacting irreversibly with the pyridoxal form of the cofactor to form a secondary amine (Rando, 1977;Avissar and Beale, 1989b). In reaction model 2, the cofactor cycles between the pyridoxamine and pyridoxal forms and gabaculine is presumably able to displace DAVA from the catalytic site of the pyridoxal-enzyme and then react with the cofactor.…”

“…In previous studies of ALA formation from glutamate, pyridoxal phosphate was added to extraction and incubation media because this compound was shown to be a cofactor of GSA aminotransferase (Avissar and Beale, 1989b;Bull et al, 1990). In preliminary experiments, the extraction and incubation media were supplemented with 20 p~ pyridoxal phosphate.…”

“…The enzyme requires pyridoxal phosphate or pyridoxamine phosphate as cofactor (Avissar and Beale, 1989b;Bull et al, 1990). …”

Biosynthesis of the Tetrapyrrole Pigment Precursor, δ-Aminolevulinic Acid, from Glutamate

“…The transamination reaction requires no added substrate or cofactor other than GSA. Native aminotransferases from barley, Chlorella and Synechocystis have mol wts of 80,000, 60,000, and 99,000, respectively (1,18). Purified aminotransferase from both barley and Synechococcus has a mol wt of 46,000 on denaturing SDS-PAGE (10).…”