Identification, Characterization, and Partial Purification of 4α-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 4α-carboxy-cholest-7-en-3β-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 Δ7-cholestenone produced and the kinetic parameters of the microsomal system have been established. 4α-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. 4α-Carboxysterol–C3-dehydrogenase/C4-decarboxylase (4α-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 4α-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 4α-methyl group to produce the 4α-carboxysterol metabolite (S. Pascal et al., J. Biol. Chem. 268, 11639, 1993) followed by oxygen-independent dehydrogenation/decarboxylation to produce an obligatory 3-ketosteroid.