Isolation and characterization of cDNA that encodes a putative mitochondrion-localizing isoform of cysteine synthase (O-acetylserine(thiol)-lyase) from Spinacia oleracea.
The cDNA clones that encode a putative mitochondrion-localizing isoform of cysteine synthase (O-acetyl-L-serine(thiol)-lyase, O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8), which is denoted as cysteine synthase C, were isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 368 amino acids containing a putative transit peptide of 30-40 amino acids at the N terminus. This leader peptide sequence exhibited several structural features common to other mitochondrion-targeting transit peptides. Homology was also detected between the putative transit peptide sequence of cysteine synthase C and other mitochondrion-targeting leader sequences. A deduced amino acid sequence of cysteine synthase C exhibited a homology of 61% with cytoplasmic isoform A and 63% with chloroplastic isoform B. A bacterial expression vector of the cDNA clone could genetically complement an Escherichia coli auxotroph lacking cysteine synthase loci and could produce the functionally active and immunoreactive cysteine synthase in E. coli. DNA blot hybridization analysis showed the presence of one or two copies of cysC gene in the genome of spinach. RNA blot hybridization analysis indicated that the expression level of cysC gene was lower than those of cysA and cysB and that the mode of cysC expression was constitutive in green and etiolated seedlings of spinach. The molecular evolutionary study of cysteine synthase proteins from plants and bacteria suggested that a common ancestor cysteine synthase gene has evolved into five cysteine synthase gene families, plant isoform A family, plant isoform B family, plant isoform C family, bacterial cysK family, and bacterial cysM family.
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
K
K
Saito
K
K
Tatsuguchi
Y
Y
Takagi
I
I
Murakoshi
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003547:Cysteine Synthase / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D007527:Isoenzymes / Q000096:biosynthesis*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology*
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D003547:Cysteine Synthase