The Non-canonical Tetratricopeptide Repeat (TPR) Domain of Fluorescent (FLU) Mediates Complex Formation with Glutamyl-tRNA Reductase

The tetratricopeptide repeat (TPR)-containing protein FLU is a negative regulator of chlorophyll biosynthesis in plants. It directly interacts through its TPR domain with glutamyl-tRNA reductase (GluTR), the rate-limiting enzyme in the formation of δ-aminolevulinic acid (ALA). Delineation of how FLU binds to GluTR is important for understanding the molecular basis for FLU-mediated repression of synthesis of ALA, the universal tetrapyrrole precursor. Here, we characterize the FLU-GluTR interaction by solving the crystal structures of the uncomplexed TPR domain of FLU (FLU^TPR) at 1.45-Å resolution and the complex of the dimeric domain of GluTR bound to FLU^TPR at 2.4-Å resolution. Three non-canonical TPR motifs of each FLU^TPR form a concave surface and clamp the helix bundle in the C-terminal dimeric domain of GluTR. We demonstrate that a 2:2 FLU^TPR-GluTR complex is the functional unit for FLU-mediated GluTR regulation and suggest that the formation of the FLU-GluTR complex prevents glutamyl-tRNA, the GluTR substrate, from binding with this enzyme. These results also provide insights into the spatial regulation of ALA synthesis by the membrane-located FLU protein.     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

In vivo functional analysis of the structural domains of FLUORESCENT (FLU)

The control of chlorophyll (Chl) synthesis in angiosperms depends on the light-operating enzyme protochlorophyllide oxidoreductase (POR). The interruption of Chl synthesis during darkness requires suppression of the synthesis of 5-aminolevulinic acid (ALA), the first precursor molecule specific for Chl synthesis. The inactivation of glutamyl-tRNA reductase (GluTR), the first enzyme in tetrapyrrole biosynthesis, accomplished the decreased ALA synthesis by the membrane-bound protein FLUORESCENT (FLU) and prevents overaccumulation of protochlorophyllide (Pchlide) in the dark. We set out to elucidate the molecular mechanism of FLU-mediated inhibition of ALA synthesis, and explored the role of each of the three structural domains of mature FLU, the transmembrane, coiled-coil and tetratricopeptide repeat (TPR) domains, in this process. Efforts to rescue the FLU knock-out mutant with truncated FLU peptides revealed that, on its own, the TPR domain is insufficient to inactivate GluTR, although tight binding of the TPR domain to GluTR was detected. A truncated FLU peptide consisting of transmembrane and TPR domains also failed to inactivate GluTR in the dark. Similarly, suppression of ALA synthesis could not be achieved by combining the coiled-coil and TPR domains. Interaction studies revealed that binding of GluTR and POR to FLU is essential for inhibiting ALA synthesis. These results imply that all three FLU domains are required for the repression of ALA synthesis, in order to avoid the overaccumulation of Pchlide in the dark. Only complete FLU ensures the formation of a membrane-bound ternary complex consisting at least of FLU, GluTR and POR to repress ALA synthesis.     

Biosynthesis of delta-Aminolevulinic Acid in Chlamydomonas reinhardtii: Study of the Transamination Mechanism Using Specifically Labeled Glutamate

The first committed intermediate of chlorophyll biosynthesis, δ-aminolevulinic acid (ALA), is synthesized from glutamate in the plant cell. The last step of ALA synthesis is a transamination reaction which converts glutamate-1-semialdehyde (GSA) to ALA. The mechanism of the transamination was examined by using glutamate, specifically labeled with either 1-¹³C or ¹⁵N, as substrate for ALA synthesis. After incubating with crude enzymes extracted from Chlamydomonas reinhardtii, the distribution of labels in purified ALA molecules was examined by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We found that both isotopes were present in the same ALA molecule. We interpret the results to mean that intermolecular transamination occurs during the conversion of GSA to ALA.     

Developmental and circadian control of the capacity for δ-aminolevulinic acid synthesis in green barley

The synthesis of δ-aminolevulinic acid (δ-ALA) is a key step in the regulation of tetrapyrrole synthesis. To study the developmentally and circadian-clock controlled mechanism that co-ordinates synthesis of chlorophylls and chlorophyll-binding proteins, δ-ALA-synthesising capacity was analysed in barley (Hordeum vulgare L.) primary leaves grown under dark/light or constant light conditions. The δ-ALA-forming activity oscillated within 24 h with a maximum at the transition of dark to light and a minimum 12 h later, indicating the involvement of the circadian oscillator during development. The capacity for δ-ALA synthesis increased transiently in the middle of barley primary leaves. The δ-ALA-forming-activity correlated well with the previously published steady-state level of mRNA for light-harvesting chlorophyll-binding proteins in space and time; this supports the view of a co-ordinate synthesis of chlorophyll and pigment-binding proteins. Steady-state levels of mRNAs encoding the three enzymes of the δ-ALA-synthesising pathway and of proteins for glutamyl-tRNA reductase (GluTR) and glutamate 1-semialdehyde aminotransferase (GSA AT; EC 5.4.3.8) were analysed for their developmental and circadian expression in barley leaves. The contents of GluTR mRNA and protein cycled parallel to the changes in δ-ALA-forming activity. The levels of GSA AT mRNA oscillated in an opposite phase, but the protein content did not show substantial oscillation under diurnal and circadian growth conditions. No circadian oscillation was detected for glutamyl tRNA synthase (GluRS; EC 6.1.1.17). Maximal GluTR mRNA content and protein was observed in the middle (segments 3 and 4) of the barley primary leaves. The developmentally controlled expression of GluTR therefore differs from that of GSA AT and GluRS, but resembles the capacity for δ-ALA synthesis in a barley leaf gradient. These data indicate that the oscillating, light-dependent and spatial expression of GluTR mRNA might contribute to the regulated formation of the chlorophyll precursor δ-ALA. Received: 29 April 1996 / Accepted 11 December 1996     

Fluorescence in blue light (FLU) is involved in inactivation and localization of glutamyl‐tRNA reductase during light exposure

Fluorescent in blue light (FLU) is a negative regulator involved in dark repression of 5‐aminolevulinic acid (ALA) synthesis and interacts with glutamyl‐tRNA reductase (GluTR), the rate‐limiting enzyme of tetrapyrrole biosynthesis. In this study, we investigated FLU‘s regulatory function in light‐exposed FLU‐overexpressing (FLUOE) Arabidopsis lines and under fluctuating light intensities in wild‐type (WT) and flu seedlings. FLUOE lines suppress ALA synthesis in the light, resulting in reduced chlorophyll content, but more strongly in low and high light than in medium growth light. This situation indicates that FLU's impact on chlorophyll biosynthesis depends on light intensity. FLU overexpressors contain strongly increased amounts of mainly membrane‐associated GluTR. These findings correlate with FLU‐dependent localization of GluTR to plastidic membranes and concomitant inhibition, such that only the soluble GluTR fraction is active. The overaccumulation of membrane‐associated GluTR indicates that FLU binding enhances GluTR stability. Interestingly, under fluctuating light, the leaves of flu mutants contain less chlorophyll compared with WT and become necrotic. We propose that FLU is basically required for fine‐tuned ALA synthesis. FLU not only mediates dark repression of ALA synthesis, but functions also to control balanced ALA synthesis under variable light intensities to ensure the adequate supply of chlorophyll.     

Light Regulation of delta-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis

Chlorophyll synthesis in Euglena, as in higher plants, occurs only in the light. The key chlorophyll precursor, δ-aminolevulinic acid (ALA), is formed in Euglena, as in plants, from glutamate in a reaction sequence catalyzed by three enzymes and requiring tRNA^Glu. ALA formation from glutamate occurs in extracts of light-grown Euglena cells, but activity is very low in dark-grown cell extracts. Cells grown in either red (650-700 nanometers) or blue (400-480 nanometers) light yielded in vitro activity, but neither red nor blue light alone induced activity as high as that induced by white light or red and blue light together, at equal total fluence rates. Levels of the individual enzymes and the required tRNA were measured in cell extracts of light- and dark-grown cells. tRNA capable of being charged with glutamate was approximately equally abundant in extracts of light- and dark-grown cells. tRNA capable of supporting ALA synthesis was approximately three times more abundant in extracts of light-grown cells than in dark-grown cell extracts. Total glutamyl-tRNA synthetase activity was nearly twice as high in extracts of light-grown cells as in dark-grown cell extracts. However, extracts of both light- and dark-grown cells were able to charge tRNA^Glu isolated from light-grown cells to form glutamyl-tRNA that could function as substrate for ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA), was approximately fourfold greater in extracts of light-grown cells than in dark-grown cell extracts. GSA aminotransferase activity was detectable only in extracts of light-grown cells. These results indicate that both the tRNA and enzymes required for ALA synthesis from glutamate are regulated by light in Euglena. The results further suggest that ALA formation from glutamate in dark-grown Euglena cells may be limited by the absence of GSA aminotransferase activity.     

Complex formation between glutamyl-tRNA reductase and glutamate-1-semialdehyde 2,1-aminomutase in Escherichia coli during the initial reactions of porphyrin biosynthesis

In Escherichia coli the first common precursor of all tetrapyrroles, 5-aminolevulinic acid, is synthesized from glutamyl-tRNA (Glu-tRNA(Glu)) in a two-step reaction catalyzed by glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM). To protect the highly reactive reaction intermediate glutamate-1-semialdehyde (GSA), a tight complex between these two enzymes was proposed based on their solved crystal structures. The existence of this hypothetical complex was verified by two independent biochemical techniques. Co-immunoprecipitation experiments using antibodies directed against E. coli GluTR and GSA-AM demonstrated the physical interaction of both enzymes in E. coli cell-free extracts and between the recombinant purified enzymes. Additionally, the formation of a GluTR.GSA-AM complex was identified by gel permeation chromatography. Complex formation was found independent of Glu-tRNA(Glu) and cofactors. The analysis of a GluTR mutant truncated in the 80-amino acid C-terminal dimerization domain (GluTR-A338Stop) revealed the importance of GluTR dimerization for complex formation. The in silico model of the E. coli GluTR.GSA-AM complex suggested direct metabolic channeling between both enzymes to protect the reactive aldehyde species GSA. In accordance with this proposal, side product formation catalyzed by GluTR was observed via high performance liquid chromatography analysis in the absence of the GluTR.GSA-AM complex.     

Accumulation of photosynthetic pigments in Larix decidua Mill. and Picea abies (L.) Karst. cotyledons treated with 5-aminolevulinic acid under different irradiation

European larch (Larix decidua Mill.) and Norway spruce [Picea abies (L.) Karst.] synthesize chlorophyll (Chl) in darkness. This paper compares Chl accumulation in 14-d-old dark-grown seedlings of L. decidua and P. abies after shortterm (24 h) feeding with 5-aminolevulinic acid (ALA). We used two ALA concentrations (1 and 10 mM) fed to cotyledons of both species in darkness and in continuous light. The dark-grown seedlings of L. decidua accumulated Chl only in trace amounts and the seedlings remained etiolated. In contrast, P. abies seedlings grown in darkness were green and had significantly higher Chl content. After ALA feeding, higher protochlorophyllide (Pchlide) content was observed in L. decidua than in P. abies cotyledons incubated in darkness. Although short-term ALA feeding stimulated the synthesis of Pchlide, Chl content did not change significantly in cotyledons incubated in darkness. The Chl accumulation in cotyledons fed with ALA was similar to the rate of Chl accumulation in the controls. Higher Chl accumulation was reported in control samples after illumination: 86.9% in L. decidua cotyledons and 46.4% in P. abies cotyledons. The Chl content decreased and bleaching occurred in cotyledons incubated with ALA in light due to photooxidation. Analyses of Chlbinding proteins (D1 and LHCIIb) by Western blotting proved differences between Chl biosynthesis in L. decidua and P. abies seedlings in the dark and in the light. No remarkable increase was found in protein accumulation (D1 and LHCIIb) after ALA application. Our results showed interspecific difference in Chl synthesis between two gymnosperms. Shortterm ALA feeding did not stimulate Chl synthesis, thus ALA synthesis was not the rate-limiting step in Chl synthesis in the dark.     

Physical and kinetic interactions between glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase of Chlamydomonas reinhardtii

In plants, algae, and most bacteria, the heme and chlorophyll precursor 5-aminolevulinic acid (ALA) is formed from glutamate in a three-step process. First, glutamate is ligated to its cognate tRNA by glutamyl-tRNA synthetase. Activated glutamate is then converted to a glutamate 1-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR) in an NADPH-dependent reaction. Subsequently, GSA is rearranged to ALA by glutamate-1-semialdehyde aminotransferase (GSAT). The intermediate GSA is highly unstable under physiological conditions. We have used purified recombinant GTR and GSAT from the unicellular alga Chlamydomonas reinhardtii to show that GTR and GSAT form a physical and functional complex that allows channeling of GSA between the enzymes. Co-immunoprecipitation and sucrose gradient ultracentrifugation results indicate that recombinant GTR and GSAT enzymes specifically interact. In vivo cross-linking results support the in vitro results and demonstrate that GTR and GSAT are components of a high molecular mass complex in C. reinhardtii cells. In a coupled enzyme assay containing GTR and wild-type GSAT, addition of inactive mutant GSAT inhibited ALA formation from glutamyl-tRNA. Mutant GSAT did not inhibit ALA formation from GSA by wild-type GSAT. These results suggest that there is competition between wild-type and mutant GSAT for binding to GTR and channeling GSA from GTR to GSAT. Further evidence supporting kinetic interaction of GTR and GSAT is the observation that both wild-type and mutant GSAT stimulate glutamyl-tRNA-dependent NADPH oxidation by GTR.     

Purification and Characterization of Glutamate 1-Semialdehyde Aminotransferase from Barley Expressed in Escherichia coli

The immediate precursor in the synthesis of tetrapyrroles is Δ-aminolevulinate (ALA). ALA is synthesized from glutamate in higher plants, algae, and certain bacteria. Glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) (GSA-AT), the third enzyme involved in this metabolic pathway, catalyzes the transamination of GSA to form ALA. The gene encoding this aminotransferase has previously been isolated from barley (Hordeum vulgare) and inserted into an Escherichia coli expression vector. We describe herein the purification of this recombinant barley GSA-AT expressed in Escherichia coli. Coexpression of GroEL and GroES is required for isolation of active aminotransferase from the soluble protein fraction of Escherichia coli. Purified GSA-AT exhibits absorption maxima characteristic of vitamin B₆-containing enzymes. GSA-AT is primarily in the pyridoxamine form when isolated and can be interconverted between this and the pyridoxal form by addition of 4,5-dioxovalerate and 4,5-diaminovalerate. The conversion of GSA to ALA under steady-state conditions exhibited typical Michaelis-Menten kinetics. Values for K_m (d,l-GSA) and k_cat were determined to be 25 micromolar and 0.11 per second, respectively, by nonlinear regression analysis. Stimulation of ALA synthesis by increasing concentrations of d,l-GSA at various fixed concentrations of 4,5-diaminovalerate supports the hypothesis that 4,5-diaminovalerate is the intermediate in the synthesis of ALA.     

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.     

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.     

delta-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins.

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C₁ of glutamate and α-ketoglutarate into the C₅ fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C₃, C₄, and C₅ of α-ketoglutarate into the C₁–C₄ (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C₁ and C₂ of glycine and C₂ and C₃ of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to ¹⁴CO₂ is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.     

Characterization of delta-Aminolevulinic Acid Formation in Soybean Root Nodules

Formation of the heme precursor δ-aminolevulinic acid (ALA) was studied in soybean root nodules elicited by Bradyrhizobium japonicum. Glutamate-dependent ALA formation activity by soybean (Glycine max) in nodules was maximal at pH 6.5 to 7.0 and at 55 to 60°C. A low level of the plant activity was detected in uninfected roots and was 50-fold greater in nodules from 17-day-old plants; this apparent stimulation correlated with increases in both plant and bacterial hemes in nodules compared with the respective asymbiotic cells. The glutamate-dependent ALA formation activity was greatest in nodules from 17-day-old plants and decreased by about one-half in those from 38-day-old plants. Unlike the eukaryotic ALA formation activity, B. japonicum ALA synthase activity was not significantly different in nodules than in cultured cells, and the symbiotic activity was independent of nodule age. The lack of symbiotic induction of B. japonicum ALA synthase indicates either that ALA formation is not rate-limiting, or that ALA synthase is not the only source of ALA for bacterial heme synthesis in nodules. Plant cytosol from nodules catalyzed the formation of radiolabeled ALA from U-[¹⁴C]glutamate and 3,4-[³H]glutamate but not from 1-[¹⁴C]glutamate, and thus, operation of the C₅ pathway could not be confirmed.