Abstract
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.
Highlights
From theFaculty of Pharmaceutical Sciences, Laboratory of Molecular Biology and Biotechnology in theResearch Center of Medicinal Resources, Chiba University, Yayoi-cho 1-33, Znage-ku, Chiba 263, Japan
The cDNA clones that encode a putative mitochon- sulfide into cysteine catalyzed by CSase is one of the key reacdrion-localizing isoformof cysteine synthase
L-serine(thio1)-lyase, 0-acetyl-L-serinaecetate-lyase(ad- catalyzes the formation of cysteinefrom 0-acetylserine and ding hydrogen sulfide),EC 4.2.99.8), which is denoted as hydrogen sulfide through the following reactions (2,3)
Summary
SCHEM1.EBiosynthetic pathway of cysteine in photosynthetic plant cells. two copy gene is expressed constitutivelyin green andetiolated spinach tissues.Molecular evolution of the CSase superfamily in plants is discussed in relation with that of the bacterial counterpart. The enzymatic activity of CSase in E. coli soluble protein extracts was determined in the reaction mixture (0.5 ml) containing 50 mM potassium phosphate, pH 8.0, 4 mM Na,S, 12.5 mM 0-acetylserine, and the enzyme solution, The incubation was performed at 30 "C for 10 min and terminated by addition ofO. ml of 7.5%trichloroaceticacid. RNA was denatured and transferred to a Hybond N+ filter with a dot-blot apparatus (Advantec Co., Tokyo) according to the standard protocol (13).Hybridization was carried out as described previously (15) For both DNA and RNAblot hybridization, the final washes of the filters were performed in 0.1 x salindsodium phosphate/ EDTA (13) and 0.1% SDSat 65 "C for 15 min
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