Biosynthesis of the cyanogenic glucosides linamarin and lotaustralin in cassava: isolation, biochemical characterization, and expression pattern of CYP71E7, the oxime-metabolizing cytochrome P450 enzyme

Abstract

Cassava (Manihot esculenta) is a eudicotyledonous plant that produces the valine- and isoleucine-derived cyanogenic glucosides linamarin and lotaustralin with the corresponding oximes and cyanohydrins as key intermediates. CYP79 enzymes catalyzing amino acid-to-oxime conversion in cyanogenic glucoside biosynthesis are known from several plants including cassava. The enzyme system converting oxime into cyanohydrin has previously only been identified in the monocotyledonous plant great millet (Sorghum bicolor). Using this great millet CYP71E1 sequence as a query in a Basic Local Alignment Search Tool-p search, a putative functional homolog that exhibited an approximately 50% amino acid sequence identity was found in cassava. The corresponding full-length cDNA clone was obtained from a plasmid library prepared from cassava shoot tips and was assigned CYP71E7. Heterologous expression of CYP71E7 in yeast afforded microsomes converting 2-methylpropanal oxime (valine-derived oxime) and 2-methylbutanal oxime (isoleucine-derived oxime) to the corresponding cyanohydrins, which dissociate into acetone and 2-butanone, respectively, and hydrogen cyanide. The volatile ketones were detected as 2.4-dinitrophenylhydrazone derivatives by liquid chromatography-mass spectrometry. A K(S) of approximately 0.9 μm was determined for 2-methylbutanal oxime based on substrate-binding spectra. CYP71E7 exhibits low specificity for the side chain of the substrate and catalyzes the conversion of aliphatic and aromatic oximes with turnovers of approximately 21, 17, 8, and 1 min(-1) for the oximes derived from valine, isoleucine, tyrosine, and phenylalanine, respectively. A second paralog of CYP71E7 was identified by database searches and showed approximately 90% amino acid sequence identity. In tube in situ polymerase chain reaction showed that in nearly unfolded leaves, the CYP71E7 paralogs are preferentially expressed in specific cells in the endodermis and in most cells in the first cortex cell layer. In fully unfolded leaves, the expression is pronounced in the cortex cell layer just beside the epidermis and in specific cells in the vascular tissue cortex cells. Thus, the transcripts of the CYP71E7 paralogs colocalize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in cassava.

Figure 1.

Biosynthesis of the Ile- and Val-derived cyanogenic glucosides lotaustralin and linamarin in cassava with emphasis on the CYP71E7-catalyzed reaction. The conversion of Ile and Val via N-hydroxyamino acids, N,N-dihydroxyamino acids, and (E)-oximes to (Z)-oximes is catalyzed by the multifunctional and dually specific CYP79D1 and CYP79D2. CYP71E7 catalyzes the conversion of the two (Z)-oximes to cyanohydrins by consecutive dehydration and C-hydroxylation reactions. Glucosylation of the cyanohydrins by a putative UDPG-glucosyltransferase (UGT) provides lotaustralin and linamarin.

Figure 2.

Carbon monoxide difference spectrum of yeast microsomes harboring CYP71E7. The Fe²⁺·CO versus Fe²⁺ difference spectrum was recorded on the microsomal fraction of yeast expressing CYP71E7.

Figure 3.

Analysis of CYP71E7-produced cyanohydrins following dissociation into ketones, derivatization, and LC-MS analysis. A, The cyanohydrins produced in the enzyme reaction mixtures were dissociated into ketones and CN⁻ at alkaline pH. B, The volatile ketones were trapped in a center well containing an acidified solution of DNPH. C, LC-MS EIC of the 2,4-dinitrophenylhydrazones of 2-butanone (EIC 253) and acetone (EIC 239). The six chromatograms shown in each panel represent assay mixtures containing buffer only (black, solid); void vector yeast microsomes, 50 μm oxime + 1 mm NADPH (light green, solid); CYP71E7-containing yeast microsomes, 10 μm oxime – NADPH (red, dotted); CYP71E7-containing yeast microsomes, 10 μm oxime + 1 mm NADPH (blue, solid); CYP71E7-containing yeast microsomes, 50 μm oxime + NADPH (magenta, solid); and CYP71E7-containing yeast microsomes, 100 μm oxime + 1 mm NADPH (dark green, dotted).

Figure 4.

Substrate-binding properties of CYP71E7 as analyzed by optical difference spectroscopy. A, Trace 1, baseline recorded with CYP71E7-harboring microsomes in the absence of substrate in both sample and reference cuvettes; traces 2, 3, and 4, spectra after the addition of ileox (0.5, 1, and 3 μm, respectively) to the sample cuvette. B, Trace 1, spectrum of CYP71E7-harboring microsomes saturated with the cytochrome P450 inhibitor n-octylamine (100 μm); trace 2, baseline recorded upon the addition of equal amounts of n-octylamine (100 μm) to CYP71E7-harboring microsomes in both sample and reference cuvettes; traces 3, 4, and 5, displacement of n-octylamine by the addition of ileox (1, 10, and 30 μm, respectively) to the sample cuvette.

Figure 5.

Cellular localization of the expression of CYP79D1 and CYP71E7 in the petiole of the first unfolded leaf using in tube in situ reverse transcription-PCR analysis of 80-μm transverse sections. The expression of the transcripts was visualized using either alkaline phosphatase-labeled (black staining) or FITC-labeled (bright and light green staining) antibodies recognizing DIG. c, Cortex; e, endodermis; l, laticifers; x, xylem. Bars = 100 μm.

Figure 6.

Cellular localization of the expression of CYP79D1 and CYP71E7 in two leaf stages, a nearly unfolded leaf and the first fully unfolded leaf, using in tube in situ reverse transcription-PCR analysis of 80-μm transverse sections. A and B, Localization of CYP79D1 and CYP71E7 expression, respectively, in a nearly unfolded upper leaf. C and D, Localization of CYP79D1 expression in an unfolded leaf. The expression of the transcripts was visualized using FITC-labeled (bright and light green staining) antibodies recognizing DIG. cp, Palisade tissue; e, epidermis; v, vascular tissue. Bars = 100 μm.