Photorespiration results from the incorporation of oxygen into ribulose-1,5-bisphosphate due to the failure of RuBisCO to properly discriminate between oxygen and carbon dioxide. This process lowers photosynthetic efficiency in that CO2 and ammonia should be re-assimilated with the concomitant consumption of both ATP and reducing power. Two recent approaches, aimed at decreasing the detrimental effects of photorespiration by introducing novel metabolic pathways into plant chloroplasts, show great promise. The goal of this work was to identify and biochemically characterize a single-gene glycolate dehydrogenase for use in further improving the synthetic pathways. Forward and reverse genetics were used to identify three candidate genes in Arabidopsis thaliana; At5g06580, At4g36400 and At4g18360. The proteins encoded by these genes were expressed in Escherichia coli, purified and characterized. Moreover, in silico analysis and the analysis of loss-of-function mutants yielded insights into the significance of these novel enzymatic activities in plant metabolism. AtD-LDH, encoded by At5g06580, is a homodimeric FAD-binding flavoprotein that catalyzes the cytochrome c- dependent oxidation of substrates. The enzyme has high activity with D- and L-lactate, D-2-hydroxybutyrate and D-glycerate, but of these only D-lactate and D-2-hydroxybutyrate are bound with high affinity. Knock-out mutants show impaired growth on medium containing methylglyoxal and D-lactate. Together, the data indicates a role for AtD-LDH in the mitochondrial intermembrane space where it oxidizes D-lactate to pyruvate in the final step of methylglyoxal detoxification. AtD-2HGDH, encoded by At4g36400, is a homodimeric FAD-binding flavoprotein. The enzyme only has activity with D-2-hydroxyglutarate and uses a synthetic electron acceptor in vitro. Metabolic analysis of knock-out mutants reveals high accumulation of D-2-hydroxyglutarate in plants exposed to long periods of extended darkness, confirming that this is the in vivo substrate for the enzyme. Co-expression analysis reveals that AtD-2HGDH is co-expressed with enzymes and transporters participating in the breakdown of lipids, branched-chain amino acids and chlorophyll, all pathways that converge in the production of propionyl-CoA. Together, the data suggest a role for AtD-2HGDH in the mitochondrial matrix where it oxidizes D-2-hydroxyglutarate, most probably originating from propionyl-CoA metabolism, to 2-oxoglutarate, using an electron transfer flavoprotein as an electron acceptor. Finally, AtGOX3, encoded by At4g18360, is a peroxisomal (S)-2-hydroxy-acid oxidase with specificity towards glycolate, L-lactate and L-2-hydroxybutyrate. AtGOX3 is almost exclusively expressed in roots where it might participate in either the metabolism of L-lactate produced during hypoxia, or glycolate produced from glycolaldehyde. In this work, the identification and thorough characterization of three novel enzymatic activities in the model plant A. thaliana are described. Moreover, novel plant metabolic pathways in which these enzymes participate were discovered. The biochemical characterization of these enzymes indicated that they are not suited for use in pathways aimed at decreasing photorespiration and thus, the search for a single-gene glycolate dehydrogenase should continue
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