CHARACTERIZATION OF MITOCHONDRIAL AND CYTOSOLIC SERINE HYDROXYMETHYLTRANSFERASES
As a folate-dependent enzyme, serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of L-serine and (6S)-H4PteGlun (tetrahydrofolate, THF) to glycine and (6S)-5,10-CH2-H4PteGlun. The glycine-forming direction of the reaction plays an essential role in all organisms by funneling one-carbon units into the folate-mediated one-carbon metabolism, which is required in nucleotide biosynthesis, amino acid metabolism, methyl group biogenesis, and vitamin metabolism; the serine-forming direction is involved in photorespiration and thus vital for C3 plants. In eukaryotes, evidences exist for different SHMT isoforms localizing in different subcellular compartments. In plants, although there is evidence for SHMT activity in cytoplasm, mitochondria and plastids, little is known about the biochemical properties and physiological significance of SHMT isoforms. Lack of such knowledge is an important problem, preventing further understanding and manipulating one-carbon fluxes in numerous metabolic pathways, such as synthesis of numerous physiologically and economically important metabolites. This knowledge is also required in the study of photorespiration which influences plant resistance to biotic and abiotic stress.In order to fill this gap in knowledge, one major project in our laboratory is to characterize all the seven putative SHMTs from Arabidopsis thaliana. Our previous work has produced promising preliminary results: cDNAs of all the seven A. thaliana SHMTs (AtSHMTs) were cloned; a HPLC-based fluorometric enzyme assays measuring glycine-forming direction was developed; and a plastid isoform has been functionally expressed and characterized.As one part of the major project, my dissertation focused on mitochondrial and cytosolic SHMTs. In this research, a novel HPLC-based fluorometric method for assaying the serine-forming direction was developed; three recombinant AtSHMTs, two from mitochondria and one from the cytosol, were functionally expressed, purified and characterized with respect to Michaelis-Menten kinetics and the impact of folate polyglutamylation; finally, two mitochondrial AtSHMTs, but not the cytosolic one, were found to display elevated catalytic activity as the enzyme concentration increased when assayed in the presence of monoglutamylated folate substrates. This effect was absent in the presence of pentaglutamylated substrates. The presented data to our knowledge provide the first evidence of folate polyglutamylation having an effect on the activity state of folate-dependent enzymes.