Identification, characterization, and partial purification of 4 alpha-carboxysterol-C3-dehydrogenase/ C4-decarboxylase from Zea mays.
A microsomal preparation from seedlings of Zea mays catalyzed the NAD+-dependent oxidative decarboxylation of several substrates, including 4alpha-carboxy-cholest-7-en-3beta-ol, synthesized according to a new procedure, giving the first in vitro evidence for this enzymatic activity in a higher plant. A GC assay has been developed to detect the Delta7-cholestenone produced and the kinetic parameters of the microsomal system have been established. 4alpha-Carboxysterol decarboxylation shows an exclusive requirement for an oxidized pyridine nucleotide, with NAD+ being more efficient than NADP+. The decarboxylation reaction is independent of molecular oxygen. 4alpha-Carboxysterol-C3-dehydrogenase/C4-decarboxylase (4alpha-CD) is a microsome-bound protein which can be efficiently solubilized by detergents, including Brij W-1 and sodium cholate. The Brij W-1-solubilized enzyme was partially purified 290-fold by a combination of DEAE anion-exchange chromatography, Cibacron blue 3GA-agarose dye chromatography, and gel permeation. The apparent molecular mass of 4alpha-CD in sodium cholate was estimated to be 45 kDa. These results support the contention that demethylation at C4 of plant sterols is composed of two separate processes: an oxygen- and NAD(P)H-dependent oxidation of the 4alpha-methyl group to produce the 4alpha-carboxysterol metabolite (S. Pascal et al., J. Biol. Chem. 268, 11639, 1993) followed by oxygen-independent dehydrogenation/decarboxylation to produce an obligatory 3-ketosteroid.
Rondet
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S
Institut de Biologie Moléculaire des Plantes, CNRS UPR 406, 28 rue Goethe, Strasbourg Cedex, 67083, France.
Taton
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M
Rahier
A
A
D002262:Carboxy-Lyases / Q000737:chemistry* / Q000302:isolation & purification*; D003653:Decarboxylation; D007202:Indicators and Reagents; D008861:Microsomes / Q000201:enzymology; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D012995:Solubility; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D007202:Indicators and Reagents; D010940:Plant Proteins; D009243:NAD; D009249:NADP; C119754:4alpha-carboxysterol-C3 dehydrogenase; D002262:Carboxy-Lyases