Simon P. Gough

tRNAGlu as a cofactor in δ-aminolevulinate biosynthesis: steps that regulate chlorophyll synthesis

Abstract In plants δ-aminolevulinate is formed from the intact carbon skeleton of glutamate catalysed by the action of three enzymes. The first step of the pathway is activation of glutamate by ligation to δ-ALA-RNA, a reaction identical to that in protein synthesis. Intriguingly, this RNA has been identified as the chloroplast tRNA Glu .

A homogenizer with replaceable razor blades for bulk isolation of active barley plastids

A modification of the cutting device of a kitchen homogenizer is described which allows the preparation of biochemically active greening barley plastids. The new cutting device consists of four easily replaceable razor blades. Intact plastids are isolated from the immature leaves of spinach or from greening barley leaves with a yield of 10% and from etiolated barley with a yield of 6%.

Biosynthesis of Δ-aminolevulinate in greening barley leaves: Glutamate 1-semialdehyde aminotransferase

L-Glutamate-1-semialdehyde was synthesized by catalytic hydrogenation of N-carbobenzoxy-L-glutamyl-1-chloride-5-benzyl ester. Soluble protein extracts of chloroplasts isolated from greening barley leaves enzymically converted L-glutamate-1-semialdehyde to δ-aminolevulinate. The enzyme was partially purified by gel filtration on a Biogel column excluding proteins larger than 500,000 daltons. The enzyme had a broad pH optimum around 8.0 and required no specific cofactors for activity. Aminooxyacetate (20mM), cycloserine (20mM), ρ-chloromercuribenzoate (0.1mM), glyoxylate (20mM) and pyridoxal phosphate (5mM) inhibited δ-aminolevulinate formation from L-glutamate-1-semialdehyde. However, β- hydroxyglutamate (1mM) a potent inhibitor of L-glutamate-U-14C conversion to δ-aminolevulinate, had no effect on L-glutamate-1-semialdehyde aminotransferase. The aminotransferase activity was eluted from the Biogel column together with the enzyme activity that converted L-glutamate-U-14C into δ-aminolevulinate. Soluble proteins prepared from etiolated plastids and mature chloroplasts of barley had a low specific activity of L-glutamate-1-semialdehyde aminotransferase compared to soluble proteins from greening plastids. It is proposed that L-glutamate-1-semialdehyde aminotransferase catalyses a part reaction in the conversion of L-glutamate to δ-aminolevulinate in greening barley plastids.

Biosynthesis of Δ-aminolevulinate in greening barley leaves. IX. Structure of the substrate, mode of gabaculine inhibition, and the catalytic mechanism of glutamate 1-semialdehyde aminotransferase

Glutamic acid 1-semialdehyde hydrochloride was synthesized and purified. Its prior structural characterization was extended and confirmed by1H NMR spectroscopy and chemical analyses. In aqueous solution at pH 1 to 2 glutamic acid 1-semialdehyde exists in a stable hydrated form, but at pH 8.0 it has a half-life of 3 to 4 min. Spontaneous degradation of the material at pH 8.0 generated some undefined condensation products, but coincidentally a significant amount isomerized to 5-aminolevulinate. At pH 6.8 to 7.0, glutamate 1-semialdehyde is sufficiently stable to permit routine and reproducible assay for glutamate 1-semialdehyde aminotransferase activity. Only about 20% of the enzyme extracted from chloroplasts was sensitive to inactivation by gabaculine with no pretreatment. However, when the enzyme was exposed to 5-aminolevulinate, levulinate or 4,5-dioxovalerate in the absence of glutamate 1-semialdehyde, it was completely inactivated by gabaculine; 4,6-dioxoheptanoate had no effect on the enzyme. These results lead to the hypothesis that the aminotransferase exists in the chloroplast in a complex with pyridoxamine phosphate, which must be converted to the pyridoxal form before it can form a stable adduct with gabaculine. We propose that the enzyme catalyzes the conversion of glutamate 1-semialdehyde to 5-aminolevulinate via 4,5-diaminovalerate.

Biosynthesis of Δ-aminolevulinate in greening barley leaves VI. Activation of glutamate by ligation to RNA

The components involved in the enzymic conversion of glutamate to δ-aminolevulinate have been separated into three fractions; a Blue Sepharose bound, a chlorophyllin-(or heme) Sepharose bound and an unbound fraction. Combination of these three fractions reconstituted δ-aminolevulinate synthesis from glutamate. Participation of a specific RNA in δ-aminolevulinate synthesis was established by isolating a homogeneous RNA from the chlorophyllin-Sepharose bound fraction and reconstituting δ-aminolevulinate synthesis in the presence of the unbound and Blue Sepharose bound fractions. The RNA involved in δ-aminolevulinate synthesis was purified by high-pressure liquid chromatography and preparative gel electrophoresis. In the presence of the Blue Sepharose bound fraction, ATP and Mg2+, glutamate bound covalently to this RNA. Co(III)-ATP-o-phenanthroline bound to the RNA and strongly inhibited glutamyl-RNA formation, whereas heme and Mg-protoporphyrin at 50 μM were only slightly inhibitory. The chlorophyllin-Sepharose bound fraction also contained two other glutamate acceptor RNAs. RNAase A and snake venom phosphodiesterase strongly reduced δ-aminolevulinate synthesis and glutamyl-RNA formation, whereas addition of DNAase or spleen phosphodiesterase was only slightly inhibitory. The RNA became sensitive to the spleen enzyme after phenol extraction of the chlorophyllin-Sepharose bound fraction. E. coli tRNAGlu orwheat germ tRNA did not reconstitute δ-aminolevulinate synthesis when combined with the Blue Sepharose bound and unbound fractions. The RNA involved in δ-aminolevulinate synthesis hybridised to a 3.9 kb Hind III Pst I restriction endonuclease fragment from the barley chloroplast genome located in the large single copy region 38 kb from the large subunit gene for RuBP carboxylase and 12 kb from the inverted repeats. Glutamate 1-semialdehyde aminotransferase was labelled during35S-incorporation into greening barley leaves but not during incorporation into isolated plastids. It is suggested that an NADPH-dependent dehydrogenase involved in the reduction of glutamyl-RNA to glutamate 1-semialdehyde is present in the Blue Sepharose bound fraction.

Induced mutations in circadian clock regulator Mat-a facilitated short-season adaptation and range extension in cultivated barley

Time to flowering has an important impact on yield and has been a key trait in the domestication of crop plants and the spread of agriculture. In 1961, the cultivar Mari (mat-a.8) was the very first induced early barley (Hordeum vulgare L.) mutant to be released into commercial production. Mari extended the range of two-row spring barley cultivation as a result of its photoperiod insensitivity. Since its release, Mari or its derivatives have been used extensively across the world to facilitate short-season adaptation and further geographic range extension. By exploiting an extended historical collection of early-flowering mutants of barley, we identified Praematurum-a (Mat-a), the gene responsible for this key adaptive phenotype, as a homolog of the Arabidopsis thaliana circadian clock regulator Early Flowering 3 (Elf3). We characterized 87 induced mat-a mutant lines and identified >20 different mat-a alleles that had clear mutations leading to a defective putative ELF3 protein. Expression analysis of HvElf3 and Gigantea in mutant and wild-type plants demonstrated that mat-a mutations disturb the flowering pathway, leading to the early phenotype. Alleles of Mat-a therefore have important and demonstrated breeding value in barley but probably also in many other day-length-sensitive crop plants, where they may tune adaptation to different geographic regions and climatic conditions, a critical issue in times of global warming.

Biosynthesis of Δ-aminolevulinate in greening barley leaves IV. Isolation of three soluble enzymes required for the conversion of glutamate to Δ-aminolevulinate

The soluble enzymes converting glutamate into δ-aminolevulinate and subsequently into uroporphyrinogen were partially purified from the stroma of greening barley plastids using Sephacryl S-300 gel filtration. By affinity chromatography employing sequentially Blue Sepharose, Matrex Gel Red A and heme-Sepharose the partially purified enzymes were separated into three fractions which together are required to catalyze the synthesis of δ-aminolevulinate from glutamate: proteins binding to Blue Sepharose, proteins binding to heme-Sepharose and run-off proteins not retained by the three columns. By analysing the characteristics of these fractions the following conclusions are reached:1.Conversion of glutamate into glutamate-1-semialdehyde in the presence of ATP, Mg2+ and NADPH requires at least two proteins, one binding to heme-Sepharose and one binding to Blue Sepharose.2.Glutamate-1-semialdehyde is converted into δ-aminolevulinate by glutamate-1-semialdehyde aminotransferase, which is not retained on the affinity columns.3.The run-off protein fraction also contains δ-aminolevulinate dehydratase and porphobilinogen deaminase.4.The heme-Sepharose bound protein(s) probably converts glutamate to glutamate-1-phosphate in the presence of ATP and Mg2+ and the Blue-Sepharose bound protein(s), glutamate-1-phosphate to glutamate-1-semialdehyde, in the presence of NADPH.

Genetic regulation of chlorophyll synthesis analyzed with mutants in barley.

Barley seedlings homozygous both for the xantha-135 and tigrina-d12 mutation accumulate magnesium protopophyrins and other precursors of chlorophyllide constitutively in darkness. The homozygous double mutant xantha-f10, tigrina-O34 produces protoporphyrin constitutively. These results provide evidence for the control of chlorophyllide synthesis in higher plants through the products of regulatory genes in the nucleus.

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

Synthesis of δ-aminolevulinate is demonstrated in stroma preparations of plastids isolated from greening barley leaves. The soluble protein fraction from developing plastids catalyzed the conversion of glutamate into δ-amino-levulinate in the presence of 0.5 mM-ATP, 0.25 mM-NADPH and 10 mM-MgCl2.

Purification and partial amino acid sequence of the glutamate 1-semialdehyde aminotransferase of barley and Synechococcus

Glutamate-1-semialdehyde aminotransferase (E.C. 5.4.3.8) was purified from barley and the cyanobacteria Synechococcus PCC 6301. The purification procedure involved serial affinity chromatography and preparative polyacrylamide gel electrophoresis under non-denaturing conditions. The aminotransferase of these two organisms showed different mobilities in non-denaturing gels. In SDS-PAGE the enzyme from both organisms migrated as a single protein with an apparent molecular weight of 46.000 Da. An antibody against the barley enzyme cross-reacted with the cyanobacterial aminotransferase. This antibody also recognized a 17 kDa peptide cleaved from the barley protein with cyanogen bromide. Amino acid sequences of the NH2-termini revealed significant homology between the eucaryotic and cyanobacterial enzyme.