A Secondary Metabolic Enzyme Functioned as an Evolutionary Seed of a Primary Metabolic Enzyme

The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an ‘evolutionary seed’ to generate an essential primary metabolic enzyme.     

Biosynthesis of delta-Aminolevulinic Acid from Glutamate in Agmenellum quadruplicatum

δ-Aminolevulinic acid accumulated in the culture medium when Agmenellum quadruplicatum strain PR-6 was incubated in the presence of levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydratase, and specifically labeled glutamate and glycine. The δ-aminolevulinic acid was purified using Dowex 50W-X8 and cleaved by periodate to yield succinic acid and formaldehyde. The distribution of radioactivity in the two fragments suggested that in blue-green algae the carbon skeleton of δ-aminolevulinic acid is derived directly from glutamate. However the possibility of the pathway of δ-aminolevulinic acid synthesis, from glycine and succinyl-coenzyme A also functioning in blue-green algae was not eliminated as uptake of glycine was minimal.     

Control of the syntheses of bacteriochlorophyll,c- and b-type cytochromes,and coproporphyrin III in Rhodopseudomonas sphaeroides mutant H5

Rhodopseudomonas sphaerodes mutant H5 lacking 5-aminolevulinic acid synthase was grown phototrophically in chemostat cultures limited by malate. Tetrapyrrole formation was limited by 5-aminolevulinic acid. With variation of dilution rates the cultures exhibited two regions of almost constant cell protein, dry weight and bacteriochlorophyll levels suggesting the formation of two physiological modifications of the strain. These modifications were further characterized by differences in the rates of 5-aminolevulinic acid consumption, the production of reserve material, the stoichiometries of 5-aminolevulinic acid consumption and bacteriochlorophyll or cytochrome production, specific bacteriochlorophyll and cytochrome contents as well as the ratio of bacteriochlorophyll protein complexes. In contrast, cellular levels of coproporphyrin II stayed almost constant over the entire range of dilution rates employed. Bacteriochlorophyll and b-type cytochrome cellular levels exhibited hyperbolic dependencies on the specific rate of 5-aminolevulinic acid consumption, and c-type cytochrome levels a signmoidal dependency. Bacteriochlorophyll cellular levels showed a biphasic dependency with half maximal saturations at 2.6 and 15.4 nmol of 5-aminolevulinic acid consumed per mg of protein and h, and maximal levels of 15.2 and 21 nmol bacteriochlorophyll per mg of protein. Cellular levels of c- and b-type cytochromes were half maximally saturated at 19.5 and 14.5 nmol 5-aminolevulinic acid consumed per mg protein and h while maximal levels were reached at 0.5 and 0.17 nmol of c- and b-type cytochromes, respectively, per mg of protein.The data suggest that within the cell bacteriochlorophyll as well as c- and b-type cytochrome units are assembled according to a defined pattern of kinetics characteristic of each group of compounds. Under otherwise constant external conditions the expression of the pattern is controlled by the rate of 5-aminolevulinic acid supply.     

Biosynthesis of δ-aminolevulinic acid in Rhodopseudomonas sphaeroides

The biosynthesis of δ-aminolevulinic acid was investigated in three strains of Rhodopseudomonas sphaeroides. A wild-type strain (NCIB 8253) possessed both δ-aminolevulinic acid synthetase and γ,δ-dioxovaleric acid transaminase in the cytoplasmic and membrane cell fractions. δ-Aminolevulinic acid synthetase activities were not detected in extracts of mutant strains H5 and H5D. However, γ,δ-dioxovaleric acid transaminase was found in the cytoplasmic and membrane fractions of these latter two strains. Strain H5 required exogenously added δ-aminolevulinic acid for growth and bacteriochlorophyll synthesis. Strain H5D did not require this compound for growth and bacteriochlorophyll synthesis. γ,δ-Dioxovaleric acid added in the growth medium did not support the growth of H5, although it was actively transported into the cells. Addition of γ,δ-dioxovaleric acid to the growth medium did not enhance the growth of either the wild-type or H5D strains. These results indicate that ALA synthetase is not required for growth and bacteriochlorophyll synthesis in H5D and that γ,δ-dioxovaleric acid is probably not an intermediate in the formation of δ-aminolevulinic acid in the strains of Rhodopseudomonas sphaeroides studied. In strain H5D another pathway may function in the formation of δ-aminolevulinic acid other than that catalyzed by δ-aminolevulinic acid synthetase or γ,δ-dioxovaleric acid transaminase.     

Control of the formation of bacteriochlorophyll,and B 875- and B 850-bacteriochlorophyll complexes in Rhodopseudomonas sphaeroides mutant strain H5

Rhodopseudomonas sphaeroides mutant H5 lacking 5-aminolevulinic acid synthase was employed to study the control of the formation of total bacteriochlorophyll as well as of the B875- and B850-bacteriochlorophyll protein complexes. The organisms were grown phototrophically in a chemostat where cell protein formation was limited by iron ions and bacteriochlorophyll by 5-aminolevulinic acid. 0.07 mol of bacteriochlorophyll was formed per mol of 5-amino-levulinic acid consumed. This stoichiometric relationship was not influenced by a twelve-fold variation in light energy flux. However, cell protein levels increased and, consequently, cellular specific bacteriochlorophyll contents decreased with increases in light energy flux. The ratio of B875- to B850-pigment protein complexes was inversely proportional to the velocity of 5-aminolevulinic acid supply (mol per cell protein and time) which in this system equals the velocity of 5-aminolevulinic acid consumption and the velocity of bacteriochlorophyll formation. Light had no direct effect on the ratio of B875- per B850-pigment complexes but an indirect effect via its control of protein formation. Changes in the ratio of the two pigment complexes resulted from the fact that significantly lower amounts of 5-aminolevulinic acid supplied per protein and time were required to saturate the system assembling the B875-complexes than that assembling the B850-complexes. The data suggest lack of light-dependent control in the formation of bacteriochlorophyll and its complexes subsequent to the 5-aminolevulinic acid pool.     

The problem of 4,5-dioxovaleric acid as a precursor of 5-aminolevulinic acid and chlorophyll in green algae

During chlorophyll biosynthesis, 4,5-dioxovaleric acid is supposed to be an intermediate between 2-oxoglutarate and 5-aminolevulinic acid. Although the occurrence of 4,5-dioxovaleric acid in cells and culture filtrates of unicellular green algae treated with levulinic acid has been previously reported, improvement of the analytical method now shows that 4,5-dioxovaleric acid does not occur in these organisms but arises due to interference of 5-aminolevulinic acid in 4,5-dioxovaleric acid analysis. The significance of 4,5-dioxovaleric acid as a free metabolite in 5-aminolevulinic acid biosynthesis must therefore be reconsidered.     

Novel inhibitors of glutamyl-tRNA(Glu) reductase identified through cell-based screening of the heme/chlorophyll biosynthetic pathway

The metabolite 5-aminolevulinic acid (ALA) is an early committed intermediate in the biosynthetic pathway of heme and chlorophyll formation. In plants, 5-aminolevulinic acid is synthesized via a two-step pathway in which glutamyl-tRNA(Glu) is reduced by glutamyl-tRNA(Glu) reductase (GluTR) to glutamate 1-semialdehyde, followed by transformation to 5-aminolevulinic acid catalyzed by glutamate 1-semialdehyde aminotransferase. Using an Escherichia coli cell-based high-throughput assay to screen small molecule libraries, we identified several chemical classes that specifically inhibit heme/chlorophyll biosynthesis at this point by demonstrating that the observed cell growth inhibition is reversed by supplementing the medium with 5-aminolevulinic acid. These compounds were further tested in vitro for inhibition of the purified enzymes GluTR and glutamate 1-semialdehyde aminotransferase as confirmation of the specificity and site of action. Several promising compounds were identified from the high-throughput screen that inhibit GluTR with an I(0.5) of less than 10 microM. Our results demonstrate the efficacy of cell-based high-throughput screening for identifying inhibitors of 5-aminolevulinic acid biosynthesis, thus representing the first report of exogenous inhibitors of this enzyme.     

Inhibition of bacteriochlorophyll synthesis in Rhodobacter sphaeroides subsp. denitrificans grown in light under denitrifying conditions.

The inclusion of nitrate or nitrite in cultures of Rhodobacter spaeroides subsp. denitrificans grown heterotrophically in light depressed the formation of bacteriochlorophyll a. The pigment biosynthesis was inhibited at the stage of the reduction of chlorophyllide (chlorin) to bacteriochlorophyllide (tetrahydroporphyrin) since 3-hydroxyethylchlorophyllide a accumulated in the culture medium. The addition of exogenous 5-aminolevulinic acid to these cultures resulted in a complete restoration of bacteriochlorophyll synthesis accompanied by the accumulation of 3-vinylbacteriopheophorbide. This indicates that under these conditions bacteriochlorophyll was formed via an alternative route, in which the reduction of chlorins to tetrahydroporphyrins precedes modifications of the C-3 side chain. The multiple forms of 5-aminolevulinic acid synthase were purified from cells grown with and without nitrate. Antibodies against these proteins were raised in rabbits and used in enzyme-linked immunosorbent assays for various forms of 5-aminolevulinic acid synthase. In denitrifying cells, the amount and activity of fraction I of the enzyme was reduced by approximately 40 and 30%, respectively. Partly active enzymes from both types of cells were activated by cystine trisulfide.     

Study of the mechanisms of uptake of 5-aminolevulinic acid derivatives by PEPT1 and PEPT2 transporters as a tool to improve photodynamic therapy of tumours

Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited (14)C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice.     

Characterization of Porphobilinogen Synthase from an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

Porphobilinogen synthase (formerly 5-aminolevulinic acid dehydratase,EC 4.2.1.24 [EC] ) was purified 7,405-fold from an aerobic photosyntheticbacterium, Erythrobacter sp. strain OCh 114. The molecular weightof the enzyme was determined to be 260,000 by Sephadex G-200gel filtration. The enzyme had a single pH optimum at 8.0 andshowed no requirement for metal ion and thiol compound for itsmaximum activity. The K_m value for 5-aminolevulinic acid was0.29 mM. 4,5-Dioxovaleric acid and levulinic acid were foundto be competitive inhibitors of the enzyme, with K_i values of0.65 and 0.80 mM, respectively. The enzyme was extremely labilein acidic pH and almost completely lost its activity within1 h at pH 6.0 and 30?C. This Erythrobacter enzyme seems to besimilar to the enzyme from the anaerobic photosynthetic bacteriumRhodobacter capsulatus in its molecular and catalytic properties. (Received February 17, 1988; Accepted May 9, 1988)     

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.     

Labeling of porphobilinogen deaminase by radioactive 5-aminolevulinic acid in isolated developing pea chloroplasts

A protein had been previously described, which was labeled by radioactive 5-aminolevulinic acid in isolated developing chloroplasts. In the present study we have shown that this protein (Mr approximately equal to 43,000) probably exists as a monomer in the chloroplast stroma. The labeling is blocked if known inhibitors of 5-aminolevulinic acid dehydratase are added to the incubation mixture, and is markedly decreased in intensity if nonradioactive 5-aminolevulinate or porphobilinogen are added to the incubation mixture; other intermediates in the porphyrin biosynthetic pathway, uroporphyrinogen III, uroporphyrin III, and protoporphyrin IX, do not decrease the labeling of the 43-kDa protein appreciably. Nondenaturing gels of the proteins isolated from the incubation with radioactive 5-aminolevulinic acid were stained for porphobilinogen deaminase activity. A series of red fluorescent bands was obtained which coincided with the radioactive bands visualized by autoradiography. It is concluded that the soluble chloroplast protein that is labeled in organello by radioactive 5-aminolevulinic acid is porphobilinogen deaminase.     

Selenium as a novel regulator of porphyrin biosynthesis in germinating seedlings of mung bean (Phaseolus vulgaris)

5-Aminolevulinic acid, porphyrin and chlorophyll contents as well the activities of 5-aminolevulinic acid dehydratase and PBG deaminase were studied in selenium treated mung bean seedlings. Selenium had no effect on 5-aminolevulinic acid synthetic ability but inhibited 5-aminolevulinic acid dehydratase and PBG deaminase activities. Further, it was observed that selenium induced accumulation of protoporphyrin-IX and Mg-protoporphyrin ester and decreased chlorophyll levels in both light and dark-grown seedlings. The results suggest the possible regulatory role of selenium on chlorophyll synthesis by interacting with sulfhydryl containing enzymes 5-aminolevulinic acid dehydratase and porphobilinogen deaminase.     

The Azorhizobium caulinodans nitrogen-fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status

The nucleotide sequence of the Azorhizobium caulinodans ORS571 nifA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen-fixing species. Highly conserved domains, including helix-turn-helix and ATP-binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5' upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifA/ntr-mediated control and a consensus element found in the 5' upstream region of the Bradyrhizobium japonicum 5-aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA-lac fusion was constructed using miniMu-lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta). NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA-lac expression.     

5-氨基乙酰丙酸的光动力应用研究进展

对光合细菌、藻类及其它细菌的5-氨基乙酰丙酸(5-ALA)产量进行了对比,类球红细菌(Rhodobacter sphaeoides)在黑暗、厌氧条件下培养,可产生大量的胞外5-ALA。5-ALA在农业生产中作为光动力除草剂、杀虫剂取得了很好效果。并且,ALA对提高植物的抗盐、抗冷冻能力也有一定作用。近年来,ALA在癌症治疗、肿瘤诊断方面也得到了广泛应用。     

13C NMR evidence for bacteriochlorophyll c formation by the C5 pathway in green sulfur bacterium, Prosthecochloris

The 13C NMR spectra of the pheophorbide of bacteriochlorophyll c, formed in the presence of L-[1-13C]glutamate and [2-13C]glycine and [13C]bicarbonate in Prosthecochloris aestaurii, were analysed. The isotope in the glutamate was specifically incorporated into the eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid, while no specific enrichment of these eight carbon atoms was observed in the spectrum of the pigment formed in the presence of [2-13C]glycine. These labelling patterns provide evidence for the operation of the C5 pathway of 5-aminolevulinic acid synthesis for bacteriochlorophyll c formation in the bacterium. The labelling of bacteriochlorophyll c by [13C]bicarbonate is consistent with its formation from 5-[1,4,5-13C]aminolevulinic acid formed by the C5 pathway from [1,2,5-13C]glutamic acid. It is proposed that this glutamate is the transamination product of 2-[1,2,5-13C]oxoglutaric acid, arising by carboxylation of [1,4-13C]succinyl-CoA with 13CO2 catalysed by 2-oxoglutaric acid synthase activity, and that the labelled succinyl-CoA is, in turn, derived by the fixation of 13CO2 by the reductive tricarboxylic acid cycle. The 13C chemical shifts of two sp2 quaternary carbons of bacteriopheophorbide c methyl ester (C-2 and C-4) were reassigned.     

13C-NMR evidence of bacteriochlorophyll a formation by the C5 pathway in Chromatium

The 13C-NMR spectra of bacteriochlorophyll a formed in the presence of L-[1-13C]glutamate and [2-13C]glycine in Chromatium vinosum strain D were analyzed. The isotope in the glutamate was specifically incorporated into eight carbon atoms in the tetrapyrrole macrocycle derived from the C-5 of 5-aminolevulinic acid (ALA), and the 13C in glycine was incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of bacteriochlorophyll a. These labeling patterns provide evidence for the exclusive operation of the C5 pathway in ALA biosynthesis in the bacterium. The 13C chemical shifts of two quaternary carbons (C-9 and C-16) of bacteriochlorophyll a were reassigned in the present study.     

Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.

6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.     

Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.