Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

• Published in: The Journal of biological chemistry Vol. 275; no. 3; pp. 1966 - 1975
• Main Authors: Andersen, M.D, Busk, P.K, Svendsen, I, Moller, B.L
• Format: Journal Article
• Language: English
• Published: United States 21.01.2000
• Subjects: amino acid metabolism; Amino Acid Sequence; amino acid sequences; biosynthesis; Blotting, Southern; Catalysis; Cells, Cultured; cloning; Cloning, Molecular; complementary DNA; cyanogenic glycosides; CYP79D1 gene; CYP79D2 gene; cytochrome P-450; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - physiology; enzyme activity; genbank/af140613; genbank/af140614; gene expression; gene transfer; genetic transformation; glucosides; Glucosides - biosynthesis; Glucosides - metabolism; isoleucine; Isoleucine - metabolism; linamarin; lotaustralin; Manihot - enzymology; Manihot - genetics; Manihot esculenta; Models, Chemical; Molecular Sequence Data; Nitriles - metabolism; nucleotide sequences; Pichia; Pichia - metabolism; Pichia pastoris; Plants, Genetically Modified - genetics; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Substrate Specificity; valine; Valine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.3.1966

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.