An O-Acetylserine (Thiol) Lyase cDNA from Spinach

Cys biosynthesis represents the essential step of incorporation of reduced sulfur into an organic compound in microorganisms and plants. OAS-TL (EC 4.2.99.8; also called Cys synthase) catalyzes the formation of L-CYS from free or bound sulfide and O-acetyl-L-Ser. Both the substrate O-acetylserine and the product Cys of this reaction are postulated to be involved in the regulation of sulfur uptake and assimilation (Giovanelli et al., 1980; Neuenschwander et al., 1991). OASTL has been purified to apparent homogeneity from spinach (Spinacia oleracea L.) leaf extracts (Murakoshi et al., 1985) and spinach chloroplasts (Droux et al., 1992) and consists of two identical subunits of 35 kD with a pyridoxal phosphate cofactor. Isoforms of OAS-TL occur in the cytosol, chloroplasts, and mitochondria of spinach leaves (Lunn et al., 1990). We report here the complete cDNA sequence for a nuclearencoded plastid isoform of OAS-TL from spinach and its expression in different organs. A spinach young leaf cDNA library was screened with a rabbit polyclonal antiserum originally generated against a soluble chloroplast protein fraction that had been fractionated for RNA binding activity by Suc gradient centrifugation and heparin affinity chromatogaphy. Four independent cDNA clones were isolated from 500,000 plaques. The sequences of the cDNA insert ends showed that the four clones differed only in length. The complete nucleotide sequence of the longest cDNA insert (designated plOAS-TL) was determined for both strands. A comparison of the predicted amino acid sequence with sequences in the GenBank/EMBL data base revealed extensive homology to the proteins encoded by the Escherichia coli and Salmonella thyphimurium cysK and cysM genes, which encode Cys synthase activity (Byme et al., 1988). Amino acid homology to OAS-TL from Capsicum annuum chromoplasts (Romer et al., 1992) is 70%, including a putative chloroplast transit sequence (von Heijne et al., 1989), and 77% for the mature proteins. The sequence deduced from plOAS-TL is consistent with the total amino acid composition as well as an amino terminal peptide sequence reported for the purified, mature OAS-TL from spinach chloroplasts (Droux et al., 1992). This sequence begins at amino acid position 52 of the plOAS-TL cDNA clone, which would indicate a mature subunit of 35.7 kD. Therefore, the cDNA