Determinants governing the CYP74 catalysis: conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis.
Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the "I-helix central domain" ("oxygen binding domain") are the primary determinants of CYP74 type of catalysis.
Toporkova
Yana Y
YY
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, 420111 Kazan, Russia.
Gogolev
Yuri V
YV
Mukhtarova
Lucia S
LS
Grechkin
Alexander N
AN
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D019295:Computational Biology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D018551:Solanum lycopersicum / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D020539:Sequence Analysis, Protein
D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C100485:divinyl ether synthase; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase