Jon D. Weinstein

Enzymatic conversion of glutamate to δ-aminolevulinate in soluble extracts of the unicellular green alga, Chlorella vulgaris

Cell-free preparations from the unicellular green alga, Chlorella vulgaris, catalyze the conversion of glutamate to delta-aminolevulinate, which is the first committed step in heme and chlorophyll biosynthesis. Most activity remains in the supernatant fraction after centrifugation at 264,000g. Additional activity can be solubilized from the high-speed pellet by treatment with 0.5 M NaCl. After gel filtration through Sephadex G-25, the reaction catalyzed by the high-speed supernatant requires glutamate, ATP, Mg2+, and NADPH. Boiled extract is inactive. The pH optimum is between 7.8 and 7.9 and the temperature optimum is 30 degrees C. Concentrations required for half-maximal activity are 0.05 mM glutamate, 0.4 mM ATP, 6 mM MgCl2, and 0.4 mM NADPH or 0.7 mM NADH. The reaction requires no additional amino donor. Involvement of pyridoxal phosphate in the catalytic mechanism is suggested by sensitivity to pyridoxal antagonists; 50% inhibition is achieved with 5 microM gabaculine or 0.4 mM aminooxyacetate. Involvement of two or more enzymes is suggested by the nonlinear reaction rate dependence on protein concentration. Evidence for the involvement of an activated glutamate intermediate was obtained by product formation after sequential addition and removal of substrates, and by inhibition (80%) with 1 mM hydroxylamine. Protoheme inhibits the activity by 50% at 1.2 microM. Preincubation of the extract with ATP causes stimulation and/or stabilization of the activity compared to preincubation without ATP or no preincubation. In preparations obtained from C. vulgaris strain C-10, which requires light for greening, dark-grown cells yield one-third as much activity as 4-h-greened cells.

RNA is required for enzymatic conversion of glutamate to δ-aminolevulinate by extracts of Chlorella vulgaris

Formation of delta-aminolevulinic acid (ALA) from glutamete catalyzed by a soluble extract from the unicellular green alga, Chlorella vulgaris, was abolished after incubation of the cell extract with bovine pancreatic ribonuclease A (RNase). Cell extract was prepared for the ALA formation assay by high-speed centrifugation and gel-filtration through Sephadex G-25 to remove insoluble and endogenous low-molecular-weight components. RNA hydrolysis products did not affect ALA formation, and RNase did not affect the ability of ATP and NADPH to serve as reaction substrates, indicating that the effect of RNase cannot be attributed to degradation of reaction substrates or transformation of a substrate or cofactor into an inhibitor. The effect of RNase was blocked by prior addition of placental RNase inhibitor (RNasin) to the cell extract, but RNasin did not reverse the effect of prior incubation of the cell extract with RNase, indicating that RNase does not act by degrading a component generated during the ALA-forming reaction, but instead degrades an essential component already present in active cell extract at the time the ALA-forming reaction is initiated. After inactivation of the cell extract by incubation with RNase, followed by administration of RNasin to block further RNase action, ALA-forming activity could be restored to a higher level than originally present by addition of a C. vulgaris tRNA-containing fraction isolated from an active ALA-forming preparation by phenol extraction and DEAE-cellulose chromatography. Bakers yeast tRNA, wheat germ tRNA, Escherichia coli tRNA, and E. coli tRNAglu type II were unable to reconstitute ALA-forming activity in RNase-treated cell extract, even though the cell extract was capable of catalyzing the charging of some of these RNAs with glutamate.

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella.

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.

Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells

Chick embryo liver cells, when cultured for 41 h in the presence of [2-14C]mevalonic acid, took up label and incorporated radioactivity into heme a, but not into protoheme. Incubation of cells with delta-[4-14C]aminolevulinic acid (ALA) resulted in uptake of label and incorporation of radioactivity into both protoheme and heme a. These results show that both protoheme and heme a are synthesized during the incubation period, and that mevalonic acid is a specific precursor of the farnesyl moiety of heme a. Incubation of cells with [1,2-14C]acetate plus N-methyl mesoporphyrin IX, an inhibitor of heme synthesis, resulted in negligible incorporation of label into protoheme and heme a, although cellular lipids were highly labeled. This result indicates that the heme purification methods employed were capable of separating hemes from lipids, and that the measured incorporation of label into hemes from [14C]mevalonic acid and [14C]ALA was not due to lipid contamination.

Chapter 5 Biochemistry and regulation of photosynthetic pigment formation in plants and algae

Publisher Summary A great diversity of naturally occurring tetrapyrroles is found among plants and algae. These species share with other organisms the need for heme-containing cytochromes and oxidases, and in addition, employ tetrapyrroles as pigments for the photosynthetic processes of trapping light energy and converting it to chemical energy. The tetrapyrrole pigments that are characteristic of plant and algal species fall into two structural groups: Mg-containing closed-macrocycle chlorophylls and their structural relatives, and open-macrocycle bilins. The chapter focuses on the biosynthesis of chlorophylls and bilins, along with a limited discussion of plant and algal hemes. The earliest well-characterized precursor that is committed to the tetrapyrrole pathway is ALA. A major branch point occurs at protoporphyrin IX, the last common intermediate leading to both the chlorophylls and the other major products. Another important branch point occurs at protoheme, which is the last common intermediate leading to both other hemes and the phycobilins (including the phytochrorne chromophore). A third branch point occurs at uroporphyrinogen III, the last common intermediate that leads to siroheme and the corrinoids.

Tetrapyrrole Precursor Biosynthesis In Euglena and Cyanidium

Two biosynthetic routes to the heme, chlorophyll (Chl), and phycobilin precursor δ-aminolevulinic acid (ALA) are known: conversion of the intact five-carbon skeleton of glutamate (Glu), and ALA synthase-catalyzed condensation of glycine (Gly) plus succinyl-CoA. Among oxygenic organisms only Euglena gracilis has yielded in vitro ALA synthase in addition to five-carbon pathway activity. The existence and physiological roles of the two pathways were assessed by determining the relative abilities of [2-14 C]Gly and [1-14 C]Glu to label protoheme, heme a, and tetrapyrrole-derived portions of Chl. In E. gracilis Chl was made predominantly from Glu, and heme a from Gly. Protoheme was made from both precursors in cells that were forming Chl, but only from Gly in dark-grown or aplastidic cells. It thus appears that E. gracilis uses ALA synthase to form mitochondrial heme precursors and the five-carbon pathway for plastid heme and Chl precursors. ALA synthase activity has not been reported in extracts of Cyanidium caldarium. Glu was incorporated to a much greater extent than Gly into both protoheme and heme a, even in cells that were unable to form Chl and phycobilins. The small incorporation of Gly could be accounted for by transfer of label to intracellular Glu pools, as determined from amino acid analysis. It thus appears that C. caldarium makes all tetrapyrroles, including mitochondrial heroes, solely from Glu.

Formation of δ-aminolevulinic Acid from Glutamic Acid in Algal Extracts: Fractionation of Activities and Biological Constraints on the RNA Requirement

The first committed step in the tetrapyrrole biosynthetic pathway, leading to chlorophylls, hemes and bilins, is the formation of 6-aminolevulinic acid (ALA). In plants, algae and some bacteria, ALA is formed from glutamate by a sequence of reactions which has been localized in the plastids in plants and eukaryotic algae (1). Soluble extracts from a variety of plants and algae are capable of converting glutamate to ALA when supplemented with ATP, Mg2+, and NADPH (2–5).

Biosynthetic Precursors of δ-Aminolevulinic Acid in Plants and Algae

The earliest universal biosynthetic precursor of the photosynthetic and respiratory tetrapyrrole pigments is the five-carbon compound δ-aminolevulinic acid (ALA). In animal cells and some bacteria, ALA has long been known to be formed by condensation of glycine and succinyl-CoA, catalyzed by the pyridoxal-requiring enzyme ALA synthase (1). Another route to ALA operates in plants, algae, and certain other cells. In this second route, ALA is formed from the intact carbon skeleton of glutamic acid. This “five-carbon” route has been found to be responsible for the biosynthesis of precursors to chlorophylls in plants (2), green algae (3), and euglenoid algae (4). This pathway is also the major or only source of tetrapyrrole precursors in bluegreen algae (5), red algae (6), some photosynthetic bacteria, including Chromatium (7) and Chlorobium (K.M. Smith and A. Huster, personal communication), and the methane producer Methanobacterium thermoautotrophicum (R.K. Thauer and H.C. Friedmann, personal communication). On the other hand, other photosynthetic bacteria are known to form ALA via the ALA synthase route (8).