Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon.
Serine acetyltransferase (SATase; EC 2.3.1.30), which catalyzes the reaction connecting serine and cysteine/methionine metabolism, plays a regulatory role in cysteine biosynthesis in plants. We have isolated a cDNA clone encoding SATase by direct genetic complementation of a Cys- mutation in Escherichia coli using an expression library of Citrullus vulgaris (watermelon) cDNA. The cDNA encodes a polypeptide of 294 amino acids (31,536 Da) exhibiting 51% homology with that of E. coli SATase. DNA-blot analysis indicated the presence of a single copy of the SATase gene (sat) in watermelon. RNA hybridization analysis suggested the relatively ubiquitous and preferential expression in the hypocotyls of etiolated seedlings. Immunoblot analysis indicated the accumulation of SATase predominantly in etiolated plants. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the SATase in an allosteric manner, indicating the regulatory function of SATase in this metabolic pathway, whereas beta-(pyrazole-1-yl)-L-alanine, a secondary metabolite formed partly through the cysteine biosynthetic pathway, showed no inhibitory effect. A multi-enzyme complex was formed from recombinant proteins of SATase and cysteine synthase (O-acetylserine(thiol)-lyase) from watermelon, suggesting efficient metabolic channeling from serine to cysteine, preventing the diffusion of intermediary O-acetyl-L-serine.
Saito
K
K
Faculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in Research Center of Medicinal Resources, Chiba University, Japan.
Yokoyama
H
H
Noji
M
M
Murakoshi
I
I
D000123:Acetyltransferases / Q000235:genetics*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000096:biosynthesis; D000598:Amino Acids, Cyclic; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003545:Cysteine / Q000096:biosynthesis*; D003547:Cysteine Synthase; D018076:DNA, Complementary / Q000235:genetics; D005638:Fruit / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D051050:Serine O-Acetyltransferase
D000596:Amino Acids; D000598:Amino Acids, Cyclic; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C045137:beta-pyrazol-1-ylalanine; D012694:Serine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; C043943:O-acetylserine; D003545:Cysteine