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In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.

The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H-protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H-protein. In the present study, we have crystallized the H-protein in its reduced state and the L-protein (lipoamide dehydrogenase or dihydrolipoamide dehydrogenase). The L-protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L-protein and the structure has been determined by X-ray crystallography. This first crystal structure of a plant dihydrolipoamide dehydrogenase is similar to other known dihydrolipoamide dehydrogenase structures. The crystal structure of the H-protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H-protein where the loaded lipoamide arm was locked into a cavity of the protein, the reduced lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L-protein. Our results strongly suggest that a direct interaction between the H- and L-proteins is not necessary for the reoxidation of the reduced lipoamide arm bound to the H-protein. This hypothesis is supported by biochemical data [Neuburger, M., Polidori, A.M., Pietre, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (2000) Eur. J. Biochem. 267, 2882-2889] and by small angle X-ray scattering experiments reported herein.

Metabolism of glycine in isolated mitochondria and protoplasts was investigated in photosynthetic, etiolated (barley and pea leaves) and fat-storing (maize scutellum) tissues using methods of [1-14C]glycine incorporation and counting of 14CO2 evolved, oxymetric measurement of glycine oxidation and rapid fractionation of protoplasts incubated in photorespiratory conditions with consequent determination of ATP/ADP ratios in different cell compartments. The involvement of different paths of electron transport in mitochondria during operation of glycine decarboxylase complex (GDC) was tested in different conditions, using aminoacetonitrile (AAN), the inhibitor of glycine oxidation in mitochondria, rotenone, the inhibitor of Complex I of mitochondrial electron transport, and inhibitors of cytochrome oxidase and alternative oxidase. It was shown that glycine has a preference to other substrates oxidized in mitochondria only in photosynthetic tissue where succinate and malate even stimulated its oxidation. Rotenone had no or small effect on glycine oxidation, whereas the role of cyanide-resistant path increased in the presence of ATP. Glycine oxidation increased ATP/ADP ratio in cytosol of barley protoplasts incubated in the presence of CO2, but not in the CO2-free medium indicating that in conditions of high photorespiratory flux oxidation of NADH formed in the GDC reaction passes via the non-coupled paths. Activity of GDC in fat-storing tissue correlated with the activity of glyoxylate-cycle enzymes, glycine oxidation did not reveal preference to other substrates and the involvement of paths non-connected with proton translocation was not pronounced. It is suggested that the preference of glycine to other substrates oxidized in mitochondria is achieved in photosynthetic tissue by switching to rotenone-insensitive intramitochrondrial NADH oxidation and by increasing of alternative oxidase involvement in the presence of glycine.

The glycine decarboxylase complex (GDC) is composed of four subunits referred to as H-, L-, P-, and T-proteins. The Saccharomyces cerevisiae GCV2 gene, encoding the P-protein has been cloned by complementation of the gsd2 mutation which prevents cells converting glycine to serine or using glycine as the sole nitrogen source. The gene, located on the right arm of chromosome XIII adjacent to TPS1, encodes a product with a M(r) of 114385. Expression of GCV2 was induced by the addition of more than 200 microM glycine in the medium, and a maximal sixfold induction occurred above 1 mM. This response was specific to glycine and was not observed for any other amino acid. Under the same conditions, the intracellular level of glycine increased up to 30-fold. The levels of P- and L-protein transcripts and GDC activity were also elevated in cells grown in the presence of glycine. Deletion analysis of the GCV2 promoter delimited the control region which contains putative regulatory sites for GCN4 and GLN3 transcription factors.

A purified extract of H-protein, a subunit of the glycine cleavage complex of the pea leaf mitochondria, was investigated by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS), using both continuous flow fast atom bombardment (CF-FAB) and electrospray ionization (ESI) mass spectrometry. Determination of the molecular weight of the entire protein, a 14 kDa subunit of the glycine decarboxylase complex, was achieved by ESI mass spectrometry and revealed covalent binding of the protein to the stabilizing agent beta-mercapto-ethanol. On-line LC/MS analysis of peptides arising from the endoproteinase Glu-C digestion of the H-protein was achieved using capillary columns (0.25 mm i.d.), and permitted confirmation of the previously reported sequence deduced from cDNA cloning experiments. The detailed interpretation of data extracted from these LC/MS experiments facilitated identification of peptides containing modified amino acid residues. In particular the identification of a lipoic acid cofactor, a rather unusual modified lysine residue which interacts with different active sites in the enzyme complex, was achieved using both LC/CF-FAB-MS and LC/ESI-MS. The exact location of this modified lysine residue was determined by obtaining fragment spectra of multiply protonated precursor ions of selected peptides, using on-line LC/MS/MS techniques.

Fractionation of carbon isotopes (13C/12C) by glycine decarboxylase (GDC) was investigated in mitochondrial preparations isolated from photosynthetic tissues of different plants (Pisum, Medicago, Triticum, Hordeum, Spinacia, Brassica, Wolffia). 20 mM glycine was supplied to mitochondria, and the CO2 formed was absorbed and analyzed for isotopic content. CO2 evolved by mitochondria of Pisum was enriched up to 8% in 12C compared to the carboxylic atom of glycine. CO2 evolved by mitochondria of the other plants investigated was enriched by 5–16 % in 13C. Carbon isotope effects were sensitive to reaction conditions (pH and the presence of GDC cofactors). Theoretical treatment of the reaction mechanism enabled us to conclude that the value and even the sign of the carbon isotope effect in glycine decarboxylation depend on the contribution of the enzyme-substrate binding step and of the decarboxylation step itself to the overall reaction rate. Therefore, the fractionation of carbon isotopes in GDC reaction was revealed which provides essential isotopic effects in plants in addition to the well-known effect of carbon isotope fractionation by the central photosynthetic enzyme, ribulose-1,5-biphosphate carboxylase.