Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.
Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach.
Saito
K
K
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Miura
N
N
Yamazaki
M
M
Hirano
H
H
Murakoshi
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003547:Cysteine Synthase / Q000235:genetics*; D015870:Gene Expression; D017343:Genes, Plant; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D014675:Vegetables / Q000235:genetics*
D010940:Plant Proteins; D012333:RNA, Messenger; D003547:Cysteine Synthase