Overexpression of a plant cysteine synthase gene and biosynthesis of a plant specific metabolite, β-(pyrazol-1-yl)-L-alanine, in Escherichia coli

Abstract

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]

α,β-Diamino Acids: Biological Significance and Synthetic Approaches

“…Willardine and isowillardine belong also to this family of heterocyclic β-substituted alanines, and in particular, willardine has been characterized as an agonist of AMPA and kainate receptors . Among others, lupinic acid, isolated from Lupinus angustifolius , β-(5-oxoisoxazol-2(5 H )-yl) alanine (BIA, β-isoxazolinone alanine), found in Lathyrus sativus , and pyrazol-1-yl alanine, characterized as a hypoglycemic agent and isolated from pressed juice of Citrullus vulgaris , have been classified within this family and have been the object of different synthetic and biosynthetic studies …”

“…A different approach takes advantage of the Michael-acceptor character of α,β-unsaturated acid derivatives to nitrogen nucleophiles. Within this context, α,β-dehydroalanine derivatives have been extremely useful starting materials containing the α-nitrogen for the Michael addition of amines. 11a, In an interesting example of the above methodology (Scheme ) from the group of Belokon, the asymmetric synthesis of α,β-diamino acids is performed via a chiral complex of Ni(II) with a dehydroalanine derivative, 250 . The presence of chiral ligand 252 in the complex provides a high facial discrimination for the attack of an amine affording the Re adduct 251 in diastereomeric ratios ranging from 90:10 to 97:3 depending on the nature of the reacting amine.…”

Cellular Biology of Sulfur and Its Functions in Plants

“…In contrast, OAS-TL over-expression approaches in cytosol and plastids increase the total cysteine content only marginally because OAS is limiting (Saito et al 1994a;Saito et al 1994b;Sirko et al 2004).…”