Formation of Mg-Containing Chlorophyll Precursors from Protoporphyrin IX, delta-Aminolevulinic Acid, and Glutamate in Isolated, Photosynthetically Competent, Developing Chloroplasts

Intact developing chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons were found to contain all the enzymes necessary for the synthesis of chlorophyllide. Glutamate was converted to Mg-protoporphyrin IX (monomethyl ester) and protoclorophyllide. δ-Aminolevulinic acid and protoporphyrin IX were converted to Mg-protoporphyrin IX, Mg-protoporphyrin IX monomethyl ester, protochlorophyllide and chlorophyllide a. The conversion of δ-aminolevulinic acid or protoporphyrin IX to Mg-protoporphyrin IX (monomethyl ester) was inhibited by AMP and p-chloromercuribenzene sulfonate. Light stimulated the formation of Mg-protoporphyrin IX from all three substrates. In the case of δ-aminolevulinic acid and protoporphyrin IX, light could be replaced by exogenous ATP. In the case of glutamate, both ATP and reducing power were necessary to replace light. With all three substrates, glutamate, δ-aminolevulinic acid, and protoporphyrin IX, the stimulation of Mg-protoporphyrin IX accumulation in the light was abolished by DCMU, and this DCMU block was overcome by added ATP and reducing power.     

(—) S-adenosyl-L-methionine-magnesium Protoporphyrin Methyltransferase, an Enzyme in the Biosynthetic Pathway of Chlorophyll in Zea mays

The enzyme (—) S-adenosyl-L-methionine-magnesium protoporphyrin methyltransferase, which catalyzes the transfer of the methyl group from (—) S-adenosyl-L-methionine to magnesium protoporphyrin to form magnesium protoporphyrin monomethyl ester, has been detected in chloroplasts isolated from Zea mays.

Zinc protoporphyrin and free protoporphyrin also act as substrates in the system, although neither one is as active as magnesium protoporphyrin.

The following scheme of chlorophyll synthesis in higher plants is proposed: δ-aminolevulinic acid → → → protoporphyrin → magnesium protoporphyrin → magnesium protoporphyrin monomethyl ester → → → chlorophyll a.

     

Purification, Characterization, and Fractionation of the delta-Aminolevulinic Acid Synthesizing Enzymes from Light-Grown Chlamydomonas reinhardtii Cells

The synthesis of δ-aminolevulinate from glutamate by Chlamydomonas reinhardtii membrane-free cell homogenates requires Mg²⁺, ATP, and NADPH as cofactors. The pH optimum is about 8.3. When analyzed by a Fractogel TSK gel filtration column the δ-aminolevulinate synthesizing enzymes, including glutamate-1-semialdehyde aminotransferase, elute with an apparent molecular weight of about 45,000. The enzymes obtained from the gel filtration column were separated into three fractions by affinity column chromatography. One fraction binds to heme-Sepharose, one to Blue Sepharose, while the enzyme converting the putative glutamate-1-semialdehyde to δ-aminolevulinic acid is retained by neither column. All three fractions are necessary for the conversion of glutamate to δ-aminolevulinate. The δ-aminolevulinate synthesizing enzymes from Chlamydomonas are sensitive to inhibition by heme but not sensitive to inhibition by protoporphyrin.     

The accumulation of bacteriochlorophyll precursors by mutant and wild-type strains of Rhodopseudomonas spheroides

1. Two mutant strains of Rhodopseudomonas spheroides, which are blocked in the synthesis of bacteriochlorophyll, accumulate pigments. These have been tentatively identified as magnesium 2,4-divinylphaeoporphyrin a₅ monomethyl ester and the magnesium derivative of 2-devinyl-2-hydroxyethyl-phaeophorbid a, formed by mutant 2/73 and 2/21 respectively. 2. Maximum extracellular production of these pigments occurs when suspensions of the organisms are incubated with low aeration in a growth medium containing iron and supplemented with glycine, succinate, methionine and Tween 80. 3. Concomitant protein synthesis is required for pigment production by the mutants from glycine and succinate but this requirement is less marked when δ-aminolaevulic acid is the substrate. 4. In the absence of Tween 80, a considerable proportion of the total pigment is retained within the cells and appears in the particulate fraction of cell-free extracts. 5. Suspensions of the parent strain containing δ-aminolaevulic acid can be made to accumulate extracellular pigments which are tentatively identified as magnesium protoporphyrin monomethyl ester and the magnesium derivative of 2-devinyl-2-hydroxyethyl-phaeophorbid a. 6. Maximum production occurs with cells incubated photosynthetically after a period of oxygen repression of bacteriochlorophyll synthesis. Formation of the phaeophorbid derivative is enhanced by 8-azaguanine or 5-fluorouracil, or by adenine deficiency in a nutritional mutant; Tween 80 is also needed and iron is essential. 7. Synthesis of bacteriochlorophyll might possibly involve the participation of lipoprotein-bound intermediates, which may be formed at the initial stage of condensation between glycine and succinyl-CoA to give δ-aminolaevulic acid.     

Succinyl-Coenzyme A Synthetase and its Role in delta-Aminolevulinic Acid Biosynthesis in Euglena gracilis

Euglena gracilis cells synthesize the key tetrapyrrole precursor, δ-aminolevulinic acid (ALA), by two routes: plastid ALA is formed from glutamate via the transfer RNA-dependent five-carbon route, and ALA that serves as the precursor to mitochondrial hemes is formed by ALA synthase-catalyzed condensation of succinyl-coenzyme A and glycine. The biosynthetic source of succinyl-coenzyme A in Euglena is of interest because this species has been reported not to contain α-ketoglutarate dehydrogenase and not to use succinyl-coenzyme A as a tricarboxylic acid cycle intermediate. Instead, α-ketoglutarate is decarboxylated to form succinic semialdehyde, which is subsequently oxidized to form succinate. Desalted extract of Euglena cells catalyzed ALA formation in a reaction that required coenzyme A and GTP but did not require exogenous succinyl-coenzyme A synthetase. GTP could be replaced with ATP. Cell extract also catalyzed glycine-and α-ketoglutarate-dependent ALA formation in a reaction that required coenzyme A and GTP, was stimulated by NADP⁺, and was inhibited by NAD⁺. Succinyl-coenzyme A synthetase activity was detected in extracts of dark- and light-grown wild-type and nongreening mutant cells. In vitro succinyl-coenzyme A synthetase activity was at least 10-fold greater than ALA synthase activity. These results indicate that succinyl-coenzyme A synthetase is present in Euglena cells. Even though the enzyme may play no role in the transformation of α-ketoglutarate to succinate in the atypical tricarboxylic acid cycle, it catalyzes succinyl-coenzyme A formation from succinate for use in the biosynthesis of ALA and possibly other products.     

An enzyme-coupled continuous spectrophotometric assay for magnesium protoporphyrin IX methyltransferases

The second committed step in chlorophyll biosynthesis is the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to magnesium protoporphyrin IX (MgP) forming MgP monomethylester (MgPME). This reaction is catalyzed by the enzyme MgP methyltransferase (ChlM). Previous investigation of this enzyme has involved the use of time-consuming techniques requiring separation of products from substrates. More recent methyltransferase studies use coupling enzymes to monitor changes in absorption/fluorescence for the measurement of activity. However, due to the spectral properties of porphyrins, many of these assays are unsuitable for analysis of the catalytic properties of ChlM. Here we report the successful development of a coupled, continuous spectrophotometric assay to measure the activity of ChlM. The product of the methyltransferase reaction, S-adenosyl-l-homocysteine (SAH), is converted into adenine and then hypoxanthine by the recombinant coupling enzymes SAH nucleosidase and adenine deaminase, respectively. The appearance of hypoxanthine results in a decrease in absorbance at 265 nm.The utility of this assay was shown by the characterization of ChlM from the cyanobacterium Synechocystis sp. PCC 6803. Kinetic parameters obtained support data acquired using the discontinuous HPLC-based assay and provide further evidence for the stimulation of ChlM by the H subunit of magnesium chelatase (ChlH).     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Reversal of alpha,alpha'-Dipyridyl-induced Porphyrin Synthesis in Etiolated and Greening Red Kidney Bean Leaves

The chemical induction of porphyrin synthesis has been investigated in etiolated and greening leaves of Phaseolus vulgaris L. var. Red Kidney. When these leaves are incubated in darkness with solutions of transition metal ion chelators such as α,α′-dipyridyl, 1,10-phenanthroline, pyridine-2-aldoxime, or other related aromatic heterocyclic nitrogenous bases, they synthesize large amounts of protochlorophyllide and Mg protoporphyrins. Greening leaves produce more porphyrin than do etiolated leaves under such conditions. If the leaves are then transferred to 1 millimolar solutions of various transition metal salts such as Fe²⁺, Zn²⁺, or Co²⁺ (but not Mn²⁺ or Mg²⁺), Mg protoporphyrin (monomethyl ester) synthesis immediately ceases and the pigment(s) rapidly disappear(s); protochlorophyllide synthesis gradually diminishes during 4 to 8 hours of treatment. The loss in Mg protoporphyrin(s) can be accounted for by a simultaneous increase in protochlorophyllide in partially greened leaves but not in etiolated leaves. In the latter, the decline in Mg protoporphyrin(s) initiated by the application of Zn²⁺ is retarded by low temperature and anaerobiosis but not by respiratory inhibitors. Cycloheximide inhibits the loss of Mg protoporphyrin(s) but does not affect their conversion to protochlorophyllide.     

Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain

The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, β, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD''s affinity for the doublet microtubule.     

Photoconversion of Photochlorophyllide in the y-1 Mutant of Chlamydomonas reinhardtii

Dark-grown y-1 mutant cells of Chlamydomonas reinhardtii accumulate protochlorophyllide (Pchlide) in both 635 nanometers (P635) and 650 nanometers (P650) forms. Plastids in these cells lack the normal thylakoid membrane structure except some remnants of membrane vesicles. Using difference spectrophotometry, P635 is shown to be photoconverted to chlorophyllide at 672 nanometers (C672) and P650 is photoconverted to C688 followed by a rapid shift to C672 (Shibata shift) and regeneration of P650. Some of the Pchlide is not photoconverted despite repeated illumination. Although P650 is destroyed by freezing and thawing, it is not transformed into P635. Freezing and thawing treatment also made Pchlide no longer photoactive.     

宽叶吊兰叶绿素生物合成的昼夜节律变化

在被子植物中,从谷氨酰-tRNA到叶绿素的生物合成是由许多酶催化的级联反应,其中间代谢产物具有较强的光反应活性和细胞毒性,因此这一过程在细胞内受到严格的调控。本研究通过检测宽叶吊兰叶片叶绿素生物合成途径的14种中间产物含量随昼夜节律的变化,探讨昼夜节律对宽叶吊兰叶绿素生物合成的影响。结果表明,中间产物ALA(δ-氨基乙酰丙酸)、PBG(胆色素原)、ProtoⅨ(原卟啉Ⅸ)、Heme(血红素)、Mg-ProtoⅨ(镁原卟啉Ⅸ)、Chlide a(叶绿素酸酯a)、Chlide b(叶绿素酸酯b)、Chl a(叶绿素a)、Chl b(叶绿素b)受光诱导,而UrogenⅢ(尿卟啉Ⅲ)、CoprogenⅢ(粪卟啉Ⅲ)和Pchlide(原叶绿素酸脂)受黑暗诱导,尤其是Pchlide在黑暗中的积累量显著增加;Mpe(镁原卟啉Ⅸ单甲酯)和Mpde(镁原卟啉Ⅸ二酯)具有2个积累峰值,分别出现在中午12∶00和夜间24∶00。说明叶绿素生物合成受昼夜节律的调控,但其中间代谢产物含量的变化规律与昼夜节律并不完全一致。     

Regulation of 5-Aminolevulinic Acid (ALA) Synthesis in Developing Chloroplasts : III. Evidence for Functional Heterogeneity of the ALA Pool

Gabaculine and 4-amino-5-hexynoic acid (AHA) up to 3.0 millimolar concentration strongly inhibited 5-aminolevulinic acid (ALA) synthesis in developing cucumber (Cucumis sativus L. var Beit Alpha) chloroplasts, while they hardly affected protochlorophyllide (Pchlide) synthesis. Exogenous protoheme up to 1.0 micromolar had a similar effect. Exogenous glutathione also exhibited a strong inhibitory effect on ALA synthesis in organello but hardly inhibited Pchlide synthesis. Pchlide synthesis in organello was highly sensitive to inhibition by levulinic acid, both in the presence and in the absence of gabaculine, indicating that the Pchlide was indeed formed from precursor(s) before the ALA dehydratase step. The synthesis of Pchlide in the presence of saturating concentrations of glutamate was stimulated by exogenous ALA, confirming that Pchlide synthesis was limited at the formation of ALA. The gabaculine inhibition of ALA accumulation occurred whether levulinic acid or 4,6-dioxohepatonic acid was used in the ALA assay system. ALA overproduction was also observed in the absence of added glutamate and was noticeable after 10-minute incubation. These observations suggest that although Pchlide synthesis in organello is limited by ALA formation, it does not utilize all the ALA that is made in the in organello assay system. Gabaculine, AHA, and probably also protoheme, inhibit preferentially the formation of that portion of ALA that is not destined for Pchlide. A model proposing a heterogenous ALA pool is described.     

Natural Killer T Cells Activated by a Lipopeptidophosphoglycan from Entamoeba histolytica Are Critically Important To Control Amebic Liver Abscess

The innate immune response is supposed to play an essential role in the control of amebic liver abscess (ALA), a severe form of invasive amoebiasis due to infection with the protozoan parasite Entamoeba histolytica. In a mouse model for the disease, we previously demonstrated that Jα18^-/- mice, lacking invariant natural killer T (iNKT) cells, suffer from more severe abscess development. Here we show that the specific activation of iNKT cells using α-galactosylceramide (α-GalCer) induces a significant reduction in the sizes of ALA lesions, whereas CD1d^−/− mice develop more severe abscesses. We identified a lipopeptidophosphoglycan from E. histolytica membranes (EhLPPG) as a possible natural NKT cell ligand and show that the purified phosphoinositol (PI) moiety of this molecule induces protective IFN-γ but not IL-4 production in NKT cells. The main component of EhLPPG responsible for NKT cell activation is a diacylated PI, (1-O-[(28∶0)-lyso-glycero-3-phosphatidyl-]2-O-(C16:0)-Ins). IFN-γ production by NKT cells requires the presence of CD1d and simultaneously TLR receptor signalling through MyD88 and secretion of IL-12. Similar to α-GalCer application, EhLPPG treatment significantly reduces the severity of ALA in ameba-infected mice. Our results suggest that EhLPPG is an amebic molecule that is important for the limitation of ALA development and may explain why the majority of E. histolytica-infected individuals do not develop amebic liver abscess.     

Effect of Dim Light on the y-1 Mutant of Chlamydomonas reinhardtii

The y-1 mutant of Chlamydomonas reinhardtii tends to die or revert to wild type when grown in the dark for a long period of time. A small amount of white light (0.5 lux) enables the y-1 mutant to grow indefinitely in a “near dark” condition. Under this condition, the y-1 mutant is physiologically and ultrastructurally similar to the dark-grown y-1 yet remains genetically stable.     

Genetic control of chlorophyll biosynthesis: Regulation of delta-aminolevulinate synthesis in Chlamydomonas

Summary A chlorophyll-deficient mutant, br _s -1, of Chlamydomonas reinhardtii has been shown to accumulate low levels of an intermediate, protoporphyrin (PROTO), and to form light-brown colonies. A double mutant, br _s -1 r-1, accumulates 15-fold more PROTO than br _s -1 and forms dark-brown colonies. Enzymes synthesizing the first intermediate of chlorophyll, delta-aminolevulinate (ALA), from these two mutants and the wild-type are equally sensitive to inhibition by heme. The activity of ALA-synthesizing enzymes from br _s -1 r-1 is similar to that of the wild-type and is more than threefold that of br _s -1. It is proposed that the ALA-synthesizing enzymes in br _s -1 are under repression while r-1 is a mutation of the regulatory gene and consequently derepresses the synthesis of its own ALA-synthesizing enzymes. In addition, by mutagenizing br _s -1, we isolated six more double mutants having the same phenotype as br _s -1 r-1. Five of them are identical to br _s -1 r-1, the remaining one (db-10) carries a second mutation nonallelic to r-1. The ALA-synthesizing enzymes from db-10 are much less sensitive to heme inhibition than those from the wild type. It is proposed that ALA synthesis in Clamydomonas is regulated both allosterically and genetically.Abbreviations PROTO protoporphyrin - ALA delta-aminolevulinate - Mg-PROTO magnesium-protoporphyrin - GSA glutamate-1-semialdehyde     

Regulation of 5-aminolevulinic Acid synthesis in developing chloroplasts : I. Effect of light/dark treatments in vivo and in organello

Intact chloroplasts isolated from greening cucumber (Cucumis sativus L. var Beit Alpha) cotyledons regenerated protochlorophyllide (Pchlide) in the dark with added cofactors from either exogenous glutamate or endogenous substrates. No other intermediates of the chlorophyll biosynthetic pathway accumulated. When inhibitors of 5-aminolevulinic acid (ALA) dehydratase were added, the Pchlide that failed to form was replaced by an excessive amount of ALA. When greening seedlings were returned to the dark, ALA-synthesizing activity in the isolated chloroplasts decreased dramatically and recovered if the dark-treated seedlings were again exposed to continuous white light prior to chloroplast isolation. Both the decline and the recovery of ALA-synthesizing activity were complete in approximately 50 minutes. Changes in chloroplast structure during in vivo light to dark and dark to light transitions (as evidenced by electron microscopy) were much slower. Exposing isolated chloroplasts from dark-treated seedlings to short white flashes before incubation transformed nearly all the endogenous Pchlide, but hardly stimulated ALA synthesis, suggesting that Pchlide does not act as a feed-back inhibitor on ALA synthesis. Chloroplasts isolated from dark-treated tissue did not form Pchlide from glutamate when incubated in the dark with added cofactors; moreover, the endogenous Pchlide did not turn over in organello. However, these chloroplasts did synthesize Pchlide from added ALA at the normal rate and synthesized ALA from glutamate at a reduced, but still significant, rate. Mg chelation was not affected by in vivo dark treatment.     

Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 × 10⁻⁶ molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and ⁵⁹Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX.