Cysteine synthase [O-acetyl-i-serine(thio1)-lyase, EC 4.2.99.81 (CSase), which is responsible for the terminal step of cysteine biosynthesis, catalyzes the formation of i-cysteine from O-acetyl-i-serine (OAS) and hydrogen sulfide. Three T-DNA vectors carrying a spinach (Spinacia oleracea) cytoplasmic CSase A cDNA (K. Saito, N. Miura, M. Yamazaki, H. Horano, 1. Murakoshi 119921 Proc Natl Acad Sci USA 89: 8078-8082) were constructed as follows: pCSK3F, cDNA driven by the cauliflower mosaic virus (CaMV) 35s RNA promoter with a sense orientation; pCSK3R, cDNA driven by the CaMV 35s promoter with an antisense orientation; pCSK4F, cDNA fused with the sequence for chloroplast-targeting transit peptide of pea ribulose-1,5-bisphosphate carboxylase small subunit driven by the CaMV 35s promoter with a sense orientation. These chimeric genes were transferred into tobacco (Nicotiana tabacum) with Agrobacterium-mediated transformation, and self-fertilized progeny were obtained. CSase activities in cell-free extracts of pCSK3F and pCSK4F transformants were 2-to 3-fold higher than those of control and pCSK3R plants. CSase activities in chloroplasts of pCSK4F transformants were severalfold higher than those of control and pCSK3F plants, indicating that the foreign CSase protein is transported and accumulated in a functionally active form in chloroplasts of pCSK4F plants. Isolated chloroplasts of a pCSK4F transformant had a more pronounced ability to form cysteine in response to addition of OAS and sulfur compounds than those of a control plant. In particular, feeding of OAS and sulfite resulted in enhanced cysteine formation, which required photoreduction of sulfite in chloroplasts. The enhanced cysteine formation in a pCSK4F plant responding to sulfite was also observed in leaf discs. In addition, these leaf discs were partially resistant to sulfite toxicity, possibly due to metabolic detoxification of sulfite by fixing into cysteine. These results suggested that overaccumulated foreign CSase in chloroplasts could modulate biosynthetic flow of cysteine in response to sulfur stress.

Comparison of seven deduced amino acid sequences of cysteme synthase ( O-acetyl-L-serine (thtol)-lyase, EC 4.2.99.8) from plants and bacterta disclosed the presence of I2 conserved Lys residues. which can be candtdates for a functional bindmg site for pyrtdoxal phosphate cofactor. These 12 conserved Lys restdues in a cDNA clone encoding spmach cysteine synthase A were replaced with Gly by oligonucleotlde-directed in vttro mutagenests.These Lys -+ Gly mutated cDNAs were transferred mto ~s~~~~~~c~~ff coit NK3, a cysteme auxotroph lacking both cysteme synthase loci, qsK and cysM. One mutant replaced at Lys-49 could not complement the cysteme requirement of NK3, whereas other mutants and wild-type clone could. No enzymatic acttvity of cysteme synthase A was detected either in the cell-free extracts of E co11 NK3 transformed with the Lys-49 mutant These results mdtcated that Lys-49 IS a functional residue for the catalytic activity of cysteme synthase. Thts Lys residue is conserved in other evoluttonarily related amino actd-metabohzmg enzymes catalyzing reactions involvmg the p-carbon of ammo acids.

This paper is dedicated to Professor Ian D. Spencer KAZUKI SAITO, REMO KANDA, MAKOTO KUROSAWA, and ISAMU MURAKOSHI. Can. J. Chem. 72, 188 (1994). Cysteine synthase (EC 4.2.99.8) in higher plants is responsible for biosynthesis of not only cysteine but also some nonprotein amino acids such as P-(pyrazol-1-y1)-L-alanine. The cDNA of a cysteine synthase from spinach (Spinacia oleracea) was inserted into pET8c (=pET3d) under the transcriptional control of strong T7 promoter to yield an overexpression vector pCEK1. The amount of the exogenous cysteine synthase was increased up to 40% of the total soluble protein of Escherichia coli transformed with pCEK1. P-(Pyrazol-1-y1)-L-alanine, a specific metabolite in plants of the Cucurbitaceae, was biosynthesized by overexpressed cysteine synthase from pyrazole in the presence of 0-acetyl-L-serine and serine, in vitro and in vivo, respectively. The present study provides the system for mechanistic investigation of biosynthesis of cysteine and biogenetically related P-substituted alanines at molecular genetic level.KAZUKI SAITO, REMO KANDA, MAKOTO KUROSAWA et ISAMU MURAKOSHI. Can. J. Chem. 72, 188 (1994).La cystkine synthase (EC 4.2.99.8) des plantes supCrieures n'est pas uniquement responsable de la biosynthkse de la cystkine, mais aussi de quelques acides aminCs non protCiniques comme la P-(pyrzol-1-y1)-L-alanine. On a insCre la cADN d'une cystkine synthase provenant d'Cpinards (Spinacia oleracea) dans le pET8c (=pET3d) sous le contrdle transcriptionnel du puissant promoteur T7 pour obtenir un vecteur de sur expression pCEK1. On a augment6 la quantitC de cystkine synthase exogbne jusqu'h 40% de la protCine soluble totale d'Escherichia coli transform6 par du pCEK1. Utilisant une cystkine synthase surexprimCe, on a biosynthktisk la P-(pyrzol-1-y1)-L-alanine, un mCtabolite spkcifique dans les cucurbitacCes, partir de pyrazole en presence de 0-acCtyl-L-sCrine et de sCrine respectivement in vitro et in vivo. L'Ctude actuelle fournit le systbme qui permet de faire l'ktude du mecanisme de biosynthkse de la cystCine et des alanines P-substituCes biogCnCtiquement apparentCes au niveau gCnktique molCculaire.[Traduit par la redaction]

Cysteine synthase plays a key role in the sulfur assimilation pathway in plant cells. The cDNA clones encoding two isoforms of this enzyme were isolated from spinach by synthetic oligonucleotide probes. The modes of expression of these two genes differed in tissues of spinach. A heterologous expression system in Escherichia coli and transgenic tobacco was made. The application of heterologous expression to modify sulfur metabolism and to produce non-protein amino acids is discussed.Abbreviation: CSase -cysteine synthase

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