Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 alpha-demethylase from maize (Zea mays) seedlings.
The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined. In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum. P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol. The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol. The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM. They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction. Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast. This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.
Taton
M
M
Département d'Enzymologie Cellulaire et Moléculaire, Institut de Botanique, Strasbourg, France.
Rahier
A
A
D002775:Cholestadienols / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008968:Molecular Conformation; D015394:Molecular Structure; D010088:Oxidoreductases / Q000378:metabolism*; D010840:Phytosterols / Q000737:chemistry; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D002775:Cholestadienols; D010840:Phytosterols; C008693:obtusifoliol; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase