Plant sterol biosynthesis: identification and characterization of higher plant delta 7-sterol C5(6)-desaturase.
Microsomes obtained from maize seedlings catalyzed the introduction of the delta5-bond into delta7-sterols to yield the corresponding delta 5,7-sterols. Enzymatic bioassay conditions have been developed for the first time for delta 7-sterol C5(6)-desaturase in photosynthetic organisms. The properties of the microsomal system have been studied and the kinetics of the desaturation reaction has been established. The desaturation reaction requires molecular oxygen and NADH. Coenzyme efficiency studies indicate that NADH is more efficient that NADPH and that in the presence of NADH, NAD+ stimulates the desaturation process but cannot sustain the reaction by itself. The desaturation is strongly inhibited by cyanide, is sensitive to 1,10-phenanthroline and to salicylhydroxamic acid, but is insensitive to carbon monoxide, suggesting the involvement of a metal ion, presumably iron, in an enzyme-bound form in the desaturating system. From a series of incubations with delta 7-sterols and other sterol analogs, the substrate specificity for desaturation was determined. Our data indicate the substrate selectivity of the C5(6)-desaturation for 4-desmethyl-delta 7-sterols. Moreover, the results show that specificity of maize C5(6)-desaturase favored delta 7-sterols possessing a C24-methylene or ethylidene substituent compared to 24-ethyl-substituted delta 7-sterols. Finally, the results demonstrate directly that during plant sterol synthesis the delta 5-bond is introduced via the sequence delta 7-sterol-->delta 5,7-sterol-->delta 5-sterol.
Taton
M
M
Département d'Enzymologie Cellulaire et Moléculaire, UPR CNRS 406, Strasbourg, France.
Rahier
A
A
D003067:Coenzymes / Q000378:metabolism; D004579:Electron Transport; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010100:Oxygen / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D013261:Sterols / Q000096:biosynthesis* / Q000737:chemistry; D013379:Substrate Specificity; D003313:Zea mays / Q000378:metabolism
D003067:Coenzymes; D013261:Sterols; C033144:lathosterol delta-5-dehydrogenase; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010100:Oxygen