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J Biol Chem, Vol. 275, Issue 3, 1966-1975, January 21, 2000

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^*

Mette Dahl Andersen^§, Peter Kamp Busk^§, Ib Svendsen^¶, and Birger Lindberg Møller^§

From the  Plant Biochemistry Laboratory, Department of Plant Biology, Royal Veterinary and Agricultural University, the ^§ Center for Molecular Plant Physiology (PlaCe), 40 Thorvaldsensvej, 1871 Frederiksberg C, Copenhagen, and the ^¶ Department of Chemistry, Carlsberg Laboratory, 10 Gamle Carlsberg Vej, 2500 Valby, Denmark

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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF140613 and AF140614.

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