Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.
Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.
Baltas
M
M
Laboratoire de Synthèse et Physico-Chimie de Molécules d'Intérêt Biologique, Université Paul-Sabatier, 118, route de Narbonne, 31062 Toulouse cedex 4, France. baltas@chimie.ups-tlse.fr
Lapeyre
C
C
Bedos-Belval
F
F
Maturano
M
M
Saint-Aguet
P
P
Roussel
L
L
Duran
H
H
Grima-Pettenati
J
J
D000214:Acyl Coenzyme A / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D015394:Molecular Structure; D063065:Organophosphonates / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D063065:Organophosphonates; D011994:Recombinant Proteins; C119167:feruloyl-CoA; C459096:sinapoyl-coenzyme A; C058644:4-coumaroyl-coenzyme A; D000445:Aldehyde Oxidoreductases; C019904:cinnamoyl CoA reductase