Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme.
Using recombinant cinnamyl alcohol dehydrogenase isoform 2 (CAD2, EC 1.1.1.195), an NADPH-dependent aromatic alcohol dehydrogenase involved in lignification in vascular plants, we have investigated the detailed steady-state kinetic mechanism of CAD2 and the role of a serine residue in determining the cofactor specificity of CAD2. Site-directed mutagenesis (S212D) and overexpression of the WT and mutant S212D forms of CAD2 in Escherichia coli, followed by kinetic studies on the purified WT and mutant proteins, confirmed the involvement of S212D in recognizing the phosphate group of NADPH and provided information on the structural requirements for NADPH specificity. From substrate kinetic patterns and product inhibition studies both WT and S212D mutant forms of CAD2 have been shown to follow rapid equilibrium random bireactant kinetics with the value of the interaction factor (alpha) for WT (0.25) being significantly less than that for S212D CAD2 (0.45). The changes in binding energy arising from the mutation on the binding of the 2'-phosphate site of the coenzyme were assessed. A marked degree of physical interaction was detected between the enzymatic binding sites of the coniferyl alcohol substrate and the 2'-phosphate binding region, which are quite distant in the three-dimensional structure. The inhibition by 2',5'-ADP and 5'-AMP was found to be weak for both WT and S212D CAD2. Strong substrate inhibition was detected for CAD2, and its implications for plant physiological studies were assessed.(ABSTRACT TRUNCATED AT 250 WORDS)
Lauvergeat
V
V
Signaux et Messages cellulaires chez les végétaux, URA CNRS 1941, Université Paul Sabatier, Toulouse, France.
Kennedy
K
K
Feuillet
C
C
McKie
J H
JH
Gorrichon
L
L
Baltas
M
M
Boudet
A M
AM
Grima-Pettenati
J
J
Douglas
K T
KT
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005052:Eucalyptus / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007527:Isoenzymes; D010636:Phenols; D011994:Recombinant Proteins; D009243:NAD; D012694:Serine; D009249:NADP; C010559:coniferyl alcohol; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase