Identification and characterization of peroxisomal glyoxylate aminotransferase from <italic>Arabidopsis thaliana</italic>.

Abstract

Aminotransferases are pyridoxal-5<super>'</super>-phosphate dependent enzymes that transfer amino groups from amino donors to amino acceptors. In plants, there are at least forty-four different aminotransferases, involved in diverse metabolic pathways ranging from nitrogen assimilation, to cofactor biosynthesis, to photorespiration. Central to the photorespiratory pathway are two non-reversible glyoxylate transaminations: a serine:glyoxylate, and a glutamate:glyoxylate aminotransferase reaction. Enzymes catalyzing these reactions have been purified and characterized from a variety of plants, but the corresponding genes were never identified. This research describes the identification and characterization of genes from <italic>Arabidopsis thaliana </italic> encoding glyoxylate aminotransferases. Arabidopsis AGT1 is a class IV aminotransferase with serine:glyoxylate aminotransferase activity. AGT1 is encoded by a single gene, transcripts of which are most abundant in green tissues, and nearly absent from roots. Inactivation of AGT1, as observed in Arabidopsis <italic>sat</italic> mutants, results in an air-sensitive conditional lethal phenotype. Serine:glyoxylate aminotransferase activity was found exclusively in Arabidopsis peroxisomes, and Arabidopsis AGT1 was imported into isolated plant peroxisomes <italic>in vitro</italic>, consistent with a solely peroxisomal localization of this protein. Kinetic analyses of recombinant Arabidopsis AGT1 indicate that its likely <italic>in vivo</italic> catalytic function is the serine:glyoxylate aminotransferase reaction. X-ray crystallographic studies of Arabidopsis AGT1 have been initiated to understand the factors governing its substrate specificity, and the molecular consequences of specific mutations. Genes encoding two alanine aminotransferase homologs with glutamate:glyoxylate aminotransferase activity were also isolated and characterized. GGT1 and GGT2 are class I aminotransferases that catalyze glutamate:glyoxylate, alanine:glyoxylate, glutamate:pyruvate, and alanine:2-oxoglutarate transaminations with physiological kinetics. <italic>GGT1</italic> and <italic>GGT2</italic> transcripts were detected in etiolated and green seedlings, suggesting that these enzymes may be active in pathways other than photorespiration. GGT1 and GGT2 appear to be peroxisomal based on sucrose gradient subcellular fractionation data, and the presence of a type 1 peroxisome targeting signal on each enzyme. Genes encoding three Arabidopsis AGT2 homologs containing putative type 1 peroxisome targeting signals were also identified, however the catalytic functions of the corresponding proteins is unknown. Arabidopsis peroxisomes thus contain at least three glyoxylate aminotransferases, AGT1, the photorespiratory serine:glyoxylate aminotransferase, and GGT1/GGT2, two putative photorespiratory alanine aminotransferases that may also be involved in other pathways.