Biosynthesis of delta-aminolevulinic acid from the intact carbon skeleton of glutamic acid in greening barley.

Abstract

The customary route in animals and bacteria for 6-aminolevulinic acid biosynthesis is from glycine and succinyl CoA, tamine were more effective in labeling the ALA (9, 10). Therefore, an alternative route of synthesis of ALA in plants appeared to be indicated. In this paper we report the labeling of specific carbon atoms of ALA by specifically labeled glutamate, clearly indicating that the five-carbon skeleton of glutamate is incorporated intact into ALA. We discuss possible biochemical routes of the labeling pattern observed. days of growth in complete darkness at 250, the terminal 80 mm of the leaves were excised with a razor blade and placed in 10 ml (25 mm diameter) beakers containing 5 ml of distilled water. Samples comprising 5 g of leaves (approximately 60 leaves) per beaker were then subjected to experimental treatment. MATERIALS AND METHODSIncubation Conditions. The leaf samples were exposed to 400 ,uW/cm2 of cool white fluorescent light as measured with a YSI model 65 radiometer (350 lux as measured with a General Electric DW58 exposure meter) at 250 for 3 hr. Then the water from the beakers was removed and replaced with incubation solutions consisting of the appropriate [14C]glutamic acid (5 Ci/mol) and, in some cases, 25 mM levulinic acid, pH adjusted to 6.5 with KOH. In the presence of levulinic acid, ALA-dehydratase [porphobilinogen synthase; b-aminolevulinate hydro-lyase (adding b-aminolevulinate and cyclizing); EC 4.2.1.24] is inhibited, thus permitting the accumulation of ALA. Total volume of the incubation solutions was 2.0 ml in all cases. The leaf samples were placed back in the same light and in the air current of a fume hood to facilitate uptake of the solution into the leaves via transpiration. After 2 hr, the incubation solutions were almost completely taken up by the leaves, and at this time the incubation was terminated by placing the leaves in stoppered plastic vials and then freezing them in powdered dry ice.

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves

“…This difference is also evident when the two precursors are examined for their ability to label ALA in vivo (3,24).…”

“…as a precursor of ALA both in detached leaves and in isolated etio-chloroplasts (3,10,24); the occurrence of alanine:DOVA transaminase in several algae (9,21,31); the cofactor requirement for the conversion of labeled KG to [14CIALA in cell-free systems (16,31); the isolation of DOVA from greening cultures of the C-2A' mutant of Scenedesmus obliquus (4,30) and, finally, the demonstration that a considerable part of the ALA formed in greening leaves of both maize and barley was obtained by removal and reinstatement of the amino group of glutamate (15). This last observation is not consistent with the suggested route through glutamate-l-semialdehyde (15).…”

Synthesis of Δ-aminolevulinic acid and chlorophyll by isolated chloroplasts

“…A prerequisite for further studies of the genetic regulation of chlorophyll biosynthesis (9,13,21) is the isolation and characterization of the enzyme(s) converting glutamate into baminolevulinate (ALA) (3,4). Recently it has been shown that ALA formation can be carried out by isolated intact immature chloroplasts of spinach (7) and cucumber (19,20).…”

Synthesis of Δ-aminolevulinate by a chloroplast stroma preparation from greening barley leaves

“…The synthesis of b-aminolevulinate (ALA) in higher plants has been actively investigated in recent years (1,2,4,6,8,9,10,11) but there is little information on the enzyme or enzymes involved, because of the difficulties encountered in obtaining active organelle-free preparations. LOHR and FRIEDMANN partially purified two enzymes from maize leaf extracts, one catalyzing the conversion ofa-ketoglutarate into dioxovalerate in the presence of NADH and the other involved in the transamination of dioxovalerate into ALA (8).…”