Sulfur assimilation in soybean ( Glycine max [L.] Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase.
A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase; EC 2.3.1.30) was isolated by screening a soybean seedling cDNA library with a (32)P-labeled expressed sequence tag. Nucleotide sequence analysis of the isolated cDNA revealed a single open-reading frame of 858 base pairs encoding a 30-kDa polypeptide. The deduced amino acid sequence of soybean SATase revealed significant homology with other plant SATases. Analysis of genomic DNA by Southern blotting indicated that SATase is encoded by a small gene family. The authenticity of the isolated SATase cDNA was confirmed by the expression of the cDNA in an Escherichia coli cysteine-auxotrophic mutant resulting in the growth of the mutant in minimal medium without cysteine. Expression of soybean SATase in E. coli resulted in the production of a 34-kDa protein that was subsequently purified by nickel-affinity column chromatography. The purified protein exhibited SATase activity, indicating that the E. coli-expressed protein is a functionally active SATase. The recombinant soybean SATase was inhibited by l-cysteine, the end product of the cysteine biosynthetic pathway. Antibodies raised against the recombinant soybean SATase cross-reacted with a 34-kDa protein from Arabidopsis leaves, but failed to detect any proteins from soybean leaves and seeds. Reverse transcriptase-polymerase chain reaction analysis indicated that SATase mRNA was expressed at low levels during soybean seed development. In comparison to Arabidopsis leaves, the SATase activity was several-fold lower in soybean leaves and seeds, suggesting that SATase is a low-abundance enzyme.
Chronis
Demosthenis
D
Department of Agronomy, University of Missouri, Columbia, MO 65211, USA.
Krishnan
Hari B
HB
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D005816:Genetic Complementation Test; D005916:Globulins / Q000235:genetics; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase; D030262:Soybean Proteins; D013025:Glycine max / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D013455:Sulfur / Q000378:metabolism*
D017931:DNA Primers; D005916:Globulins; D007527:Isoenzymes; D010940:Plant Proteins; D030262:Soybean Proteins; D013455:Sulfur; C011474:glycinin; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase