Evidence for identity of beta-pyrazolealanine synthase with cysteine synthase in watermelon: formation of beta-pyrazole-alanine by cloned cysteine synthase in vitro and in vivo.
The responsibility of cysteine synthase (EC 4.2.99.8) from watermelon (Citrullus vulgaris) for the formation of beta-(pyrazole-1-yl)-L-alanine, a non-protein amino acid specifically accumulated in Curcubitaceae plants, was confirmed in vitro and in vivo by the cloned cDNA on expression vectors, pCCS11 and pCEN1. The cDNA sequence derived from pCCS11, an expression vector driven by the lacZ promoter, was placed under the transcriptional control of strong T7 promoter of pET3d to yield an over-expression vector, pCEN1, in Escherichia coli. The concentration of the exogenous cysteine synthase protein was increased up to approximately 10% of the total soluble protein of E. coli cells by the expression of cDNA on pCEN1. beta-(Pyrazole-1-yl)-L-alanine was formed in vitro from O-acetyl-L-serine and pyrazole by the action of cysteine synthase expressed in E. coli carrying pCCS11 or pCEN1. To confirm the responsibility of cysteine synthase for the formation of beta-(pyrazole-1-yl)-L-alanine in vivo, the feeding experiments of pyrazole and serine or O-acetyl-L-serine were carried out using the transformed E. coli culture. beta-(Pyrazole-1-yl)-L-alanine was produced in vivo by feeding the substrates to the culture of E. coli carrying pCEN1. These results provide the confirming evidence that the cloned cysteine synthase of watermelon catalyzes the formation of beta-(pyrazole-1-yl)-L-alanine, indicating that beta-pyrazolealanine synthase is identical with cysteine synthase in Cucurbitaceae plants.
Noji
M
M
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Murakoshi
I
I
Saito
K
K
D001483:Base Sequence; D003001:Cloning, Molecular; D003547:Cysteine Synthase / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli; D005638:Fruit / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D003547:Cysteine Synthase