RUNNING HEAD: CYP71E7: The oxime-metabolizing P450 in ...4 ABSTRACT Cassava (Manihot esculenta...

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RUNNING HEAD: CYP71E7: The oxime-metabolizing P450 in Manihot esculenta Crantz,

cassava

CORRESPONDING AUTHOR: Søren Bak, [email protected]

ADDRESS: Plant Biochemistry Laboratory, Department of Plant Biology and

Biotechnology, VKR Research Centre “Pro-Active Plants”, Faculty of Life Sciences,

University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen,

Denmark

TELEPHONE: +45 3533 3346

FAX: +45 3533 3333

JOURNAL RESEARCH AREA: Biochemical Processes and Macromolecular Structures

Plant Physiology Preview. Published on November 2, 2010, as DOI:10.1104/pp.110.164053

Copyright 2010 by the American Society of Plant Biologists

www.plantphysiol.orgon March 1, 2020 - Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved.

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TITLE: Biosynthesis of the cyanogenic glucosides linamarin and lotaustralin in cassava

(Manihot esculenta Crantz): Isolation, biochemical characterization and expression pattern of

CYP71E7, the oxime-metabolizing cytochrome P450 enzyme

AUTHORS: Kirsten Jørgensen, Anne Vinther Moranta, Marc Morantb, Niels Bjerg Jensen,

Carl Erik Olsen, Rubini Kannangara, Mohammed Saddik Motawia, Birger Lindberg Møller

and Søren Bak.

ADDRESS: Plant Biochemistry Laboratory, Department of Plant Biology and

Biotechnology, VKR Research Centre “Pro-Active Plants” (A.V.M., M.M., K.J., N. B. J,

M.S.M., B.L.M. and S.B.), Center for Synthetic Biology (K.J., M.S.M., B.L.M. and S.B) and

Department of Basic Sciences and Environment (C.E.O.), University of Copenhagen, 40

Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark



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FOOTNOTES

FINANCIAL SOURCES: This work has been supported by grants from the Danish

International Development Agency (DANIDA), by the Danish Research Council for

Technology and Production (FTP), the Danish National Research Foundation, the Villum

Foundation and from the UNIK Synthetic Biology Research Initiative, funded by the Danish

Ministry of Science, Technology and Innovation.

a) Current address: H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark

b) Current address: Novozymes A/S Krogshøjvej 36, 2880 Bagsværd Denmark

CORRESPONDING AUTHOR: Søren Bak, email: [email protected], fax: +45 3533 3346



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ABSTRACT

Cassava (Manihot esculenta Crantz) is a eudicotyledonous plant, which 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 M. esculenta. The enzyme system converting oxime into

cyanohydrin has previously only been identified in the monocotyledonous plant Sorghum

bicolor Moench. Using this S. bicolor CYP71E1 sequence as a query in a BLASTp search, a

putative functional homologue which exhibited a ~50% amino acid sequence identity was

found in M. esculenta. The corresponding full length cDNA clone was obtained from a

plasmid library prepared from M. esculenta 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 hydrogen cyanide

and and acetone and 2-butanone, respectively. The volatile ketones were detected as 2.4-

dinitrophenylhydrazone derivatives by LC-MS. A KS of ~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 conversion of aliphatic and aromatic oximes

with turnovers of ~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 ~90% amino acid sequence identity. In tube in situ PCR

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 co-localize with CYP79D1 and CYP79D2. We conclude that CYP71E7 is

the oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in M. esculenta.



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INTRODUCTION

Manihot esculenta Crantz (cassava) is one of the most important root crops in the world.

The drought tolerance of M. esculenta, combined with a high yield on poor soils and the

possibility to leave the starch rich tubers in the soil for extended time periods as a food and

thus harvest on demand, renders M. esculenta a key staple food especially in Africa (Nweke

et al., 2002). All parts of the M. esculenta plant contain the valine- and isoleucine-derived

cyanogenic glucosides, linamarin and lotaustralin in an approximately 97 to 3 ratio

(Lykkesfeldt and Møller, 1994). The cyanogenic glucosides are primarily synthesized in the

shoot apex (Andersen et al., 2000) and transported to the tubers where they accumulate up to

1.5 g/kg dry weight (Bokanga, 1994; Jørgensen et al., 2005a). In planta, cyanogenic

glucosides and specific β-glucosidases able to cleave the β-glucosidic linkage are

compartmentalized into different tissues or subcellular compartments including the apoplast

(Morant et al., 2008). This provides a two-component system which is activated upon cellular

disruption e.g. during food processing or by chewing insects. The cyanohydrin formed by the

action of the β-glucosidase subsequently dissociates into a ketone and toxic HCN.

The presence of cyanogenic glucosides in cassava poses a major nutritive drawback in

rural areas where cassava constitutes the sole staple crop available. Ingestion of incompletely

processed cassava derived products in combination with an unbalanced diet deficient in

sulphur amino acids may give rise to chronic cyanide intoxication, as sulphur amino acids are

required for cyanide detoxification (Rosling, 1994). In extreme cases, this may result in

severe diseases such as tropical ataxic neuropathy and konzo (Banea-Mayambu et al., 1997;

Oluwole et al., 2000). Acute cyanide intoxication inactivates the mitochondrial cytochrome

aa3 oxidase and thereby blocks cellular respiration (Nelson, 2006). Unfortunately, careful

processing of cassava tubers to remove the cyanide generating constituents results in

concomitant loss of proteins, vitamins and minerals and thus significantly reduces the

nutritional value of this important crop (Maziya-Dixon et al., 2007).

The enzymes and corresponding genes for the entire cyanogenic glucoside biosynthetic

pathway have only been identified in the monocotyledonous crop Sorghum bicolor Moench

(great millet; Jones et al., 1999). S. bicolor contains the aromatic cyanogenic glucoside

dhurrin. The dhurrin pathway is genetically simple, as only three structural genes encode the

entire pathway. Two membrane bound, multifunctional cytochromes P450, CYP79A1 and

CYP71E1, and a soluble UDPG-glucosyltransferase (UGT), UGT85B1, catalyze the

conversion of tyrosine into dhurrin (Halkier et al., 1995; Bak et al., 1998; Jones et al., 1999).



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CYP79A1 catalyzes the conversion of the amino acid tyrosine into (Z)-p-hydroxyphenyl

acetaldoxime in a reaction sequence that involves two consecutive N-hydroxylations, a

dehydration and a decarboxylation reaction, and a final isomerization reaction (Halkier et al.,

1995; Sibbesen et al., 1995). The next enzyme in the pathway, CYP71E1, converts (Z)-p-

hydroxyphenyl acetaldoxime into p-hydroxymandelonitrile via a dehydration and a C-

hydroxylation reaction (Halkier et al., 1989; Kahn et al., 1997; Bak et al., 1998). Finally, the

p-hydroxymandelonitrile is glucosylated by UGT85B1 to yield dhurrin (Jones et al., 1999).

The biosynthetic pathway is known to proceed as a highly channelled process (Møller and

Conn, 1980), and the three biosynthetic enzymes are thought to be organized within a

multienzyme complex, a metabolon (Nielsen and Møller, 2005; Nielsen et al., 2007), to

secure highly efficient conversion of the parent amino acid into the cyanogenic glucoside and

to facilitate channelling of toxic and reactive intermediates (reviewed in Jørgensen et al.,

2005b and Winkel, 2004).

Enzymes of the CYP79 family catalyze the first and rate limiting steps in cyanogenic

glucoside biosynthesis and have been identified from several cyanogenic plants (reviewed in

Bak et al., 2006) including M. esculenta (Andersen et al., 2000). CYP79 enzymes are clearly

evolutionarily conserved between monocotyledons and eudicotyledons and can be easily

distinguished from other cytochromes P450 by unique amino acid substitutions in the

otherwise generally conserved heme binding domain and in the so-called “PERF” motif in

the meander region (Bak et al., 2006, Paquette et al., 2009). CYP79 enzymes also catalyze

the first committed step in biosynthesis of the crucifer specific aliphatic, aromatic and indole

glucosinolates and camalexin (Rauhut and Glawischnig 2009). The general assumption is that

the glucosinolate pathway has evolved from the ancient cyanogenic glucoside pathway, and

that the amino acid to oxime step is catalyzed by orthologous enzymes and the two pathways

branch at the oxime (Bak et al., 2005). In addition, the CYP79 biochemical activity is readily

detected using radio labeled amino acids as a substrate (Andersen et al., 2000). In contrast,

identification of candidate CYP71E1 functional homologues or orthologs that catalyze the

oxime to cyanohydrin step remains a major challenge because of multiple gene duplications

and rearrangements resulting in numerous neo- and sub-functionalizations within the CYP71

family. This is exemplified by the presence of 52 members of the CYP71 family in the

diploid eudicotyledon Arabidopsis thaliana (thale cress), covering only two subfamilies,

CYP71A and CYP71B, with amino acid sequence identities above 55% in each subfamily

(Paquette et al., 2000; Nelson et al., 2004, Paquette et al., 2009). In addition, CYP71E1 lacks

obvious unique substitutions in otherwise conserved motifs in the amino acid sequence.



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Identification of a functional CYP71E1 homologue from plants producing aliphatic

cyanogenic glucosides is further complicated by the lack of a sensitive method to detect the

volatile reaction products. The lack of known CYP71E1 functional homologues raises the

question of whether the oxime-metabolizing enzyme is evolutionarily conserved between

monocotyledons and eudicotyledons as well, or if the post oxime step is a case of convergent

rather than divergent evolution.

In M. esculenta, the committed step from amino acid to oxime in lotaustralin and

linamarin biosynthesis is catalyzed by the isoenzymes CYP79D1 and CYP79D2 (Fig 1;

Andersen et al., 2000). These two enzymes are dually specific in that they catalyze the

conversion of both isoleucine and valine into the corresponding oximes. The location of

CYP79D1 and CYP79D2 transcripts has been examined in the first fully unfolded leaf and its

petiole in the shoot tip of two month old M. esculenta plants. CYP79D1 and CYP79D2 are

co-expressed in all tissues examined with high expression in the outer cortex, endodermis and

pericycle cell layers and in tissues surrounding laticifers, xylem and phloem cells in the

petiole (Jørgensen et al., 2005a). The presence of two apparently functional redundant CYP79

homeologs most likely reflects that M. esculenta is allopolyploid (Fregene et al., 1997).

In this study we report the identification, biochemical characterization, and expression

pattern in M. esculenta petioles and young leaves of a bifunctional P450 enzyme designated

CYP71E7, which catalyzes the conversion of isoleucine- and valine-derived oximes into their

corresponding cyanohydrins in the biosynthesis of the cyanogenic glucosides lotaustralin and

linamarin in M. esculenta. This demonstrates that the oxime-metabolizing step is conserved

in cyanogenic glucoside biosynthesis in monocotyledons and eudicotyledons.



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RESULTS

Isolation and heterologous expression of CYP71E7 cDNA

The biosynthetic pathway for the cyanogenic glucosides lotaustralin and linamarin in M.

esculenta is illustrated in Figure 1 (Andersen et al., 2000; Koch et al., 1995). The enzyme

system(s) catalyzing conversion of the aliphatic oximes (Z)-2-methylbutanal oxime (ileox)

and (Z)-2-methylpropanal oxime (valox) into the corresponding cyanohydrins has remained

elusive. In S. bicolor, the CYP71E1 enzyme has been shown to catalyze conversion of the

aromatic oxime p-hydroxyphenyl acetaldoxime (tyrox) into the corresponding cyanohydrin,

p-hydroxymandelonitrile (Bak et al., 1998). Using the S. bicolor CYP71E1 amino acid

sequence as a query in a BLASTp search, an M. esculenta sequence with ~50% identity and

~68% similarity at the amino acid level was identified (Zhang et al., 2003; AY217351).

Phylogenetic analysis grouped the M. esculenta cytochrome P450 sequence with the

CYP71E1 sequence from S. bicolor as well as with cytochrome P450 sequences from rice,

wheat and sugarcane (data not shown), four other species known to synthesize cyanogenic

glucosides (Jones, 1998). Based on the cytochrome P450 nomenclature rules

(http://drnelson.utmem.edu/CytochromeP450.html), the M. esculenta P450 sequence has been

assigned as CYP71E7. Analysis of the genome sequence of the M. esculenta line AM-560-2

(Cassava Genome Project 2009, http:://www.phytozome.net/cassava), revealed the presence

of a CYP71E7 paralog on scaffold 02340 located within 12.000 bp of CYP71E7. The two

paralogs are 90% identical and 94% similar on the amino acid level, and the ORFs are 92%

identical.

To investigate whether CYP71E7 is the M. esculenta oxime-metabolizing enzyme, a

CYP71E7 cDNA was isolated by PCR from a cDNA library prepared from top shoots of M.

esculenta. This library had previously provided CYP79D1 and CYP79D2 (Andersen et al.,

2000) encoding the multifunctional isoenzymes CYP79D1 and CYP79D2 which catalyze the

first committed and rate limiting step in the pathway (Fig. 1; Andersen et al., 2000).

Recombinant CYP71E7 was produced in Saccharomyces cerevisiae WAT11 yeast cells that

co-express the A. thaliana NADPH:cytochrome P450 reductase, ATR1 (At4g24520). ATR1 is

a diflavin protein catalyzing electron transfer from NADPH to the heme iron during the P450

reaction cycle (Pompon et al., 1996, Jensen and Møller 2010, Laursen et al., 2010). Isolated

yeast microsomes harboring recombinant CYP71E7 produced the characteristic Soret peak at

450 nm upon CO binding (Fig. 2). This indicated that the heme group was correctly



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positioned in the active site and that CYP71E7 was produced in a correctly folded and active

form.

CYP71E7 is the oxime-metabolizing cytochrome P450 in biosynthesis of lotaustralin

and linamarin

Yeast microsomes harboring CYP71E7 were assayed for their ability to convert ileox and

valox into the corresponding cyanohydrins, 2-hydroxy-2-methylbutyronitrile and acetone

cyanohydrin. The design of the assay was based on dissociation of the labile cyanohydrins

formed into hydrogen cyanide and ketones by alkalinization of the reaction mixture at the end

of the incubation period (Fig. 3A) and subsequent trapping of the volatile ketones (2-

butanone and acetone) as 2,4-dinitrophenylhydrazones (Fig. 3B). After extraction, the 2,4-

dinitrophenylhydrazones formed were identified and quantified by LC-MS. To reduce

background levels due to contaminating aldehydes and ketones in the surrounding air and to

retain the 2-butanone and acetone produced, incubations were carried out in closed glass vials

with an acidified solution of 2,4-dinitrophenylhydrazine placed in a centre well.

In the presence of NADPH and oxygen, CYP71E7 converted ileox into 2-hydroxy-2-

methylbutyronitrile with a turnover of 17 ± 1 min-1 and a Km of 21 ± 11 µM. In agreement

with this, product formation was detected already at 10 μM ileox and increased with substrate

concentration up to 100 μM ileox (Fig. 3C, EIC 253). In the absence of NADPH or in assay

mixtures using microsomes isolated from yeast transformed with an empty expression vector,

hydrazone formation above background was not observed (Fig. 3C, EIC 253). The 2,4-

dinitrophenylhydrazone assay enables detection of as little as 50 pmol 2-butanone with

negligible background interference (Fig. 3C, EIC 253). The 2,4-dinitrophenylhydrazone of 2-

butanone was detected as two components with the main component (83%) eluting at 10.6

min and representing the (E)-isomer and the minor (17%) at 9.8 min the (Z)-isomer (Figure

3C, EIC 253).

CYP71E7 catalyzed conversion of valox into acetone cyanohydrin at a turnover of 21 ± 2

min-1 (Table 1). The acetone obtained by dissociation of the acetone cyanohydrin produced a

2,4-dinitrophenylhydrazone that eluted as a single component at 6.4 min (Fig. 3C, EIC 239)

as the carbonyl functional group of acetone carries two identical substituents. As for ileox,

product formation was detected already at a substrate concentration of 10 μM (Fig. 3C, EIC

239). Acetone is omnipresent in air and absorbed to glassware in sufficient amounts to

interfere with this highly sensitive assay. Accordingly, a high and slightly variable

background level was observed even in blank samples including only buffer (Fig. 3C, EIC



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239). The variable background level prevented calculation of an accurate Km for valox. In

addition to metabolizing the two aliphatic oximes, CYP71E7 was also able to convert tyrox

and the phenylalanine-derived phenylacetaldoxime (pheox) into the corresponding

cyanohydrins, albeit with lower turnovers 8.1 ± 0.3 and 1.3 ± 0.2 min-1, respectively.

Spectral analysis of substrate binding to CYP71E7

Binding of substrates within the active site of cytochromes P450 induces spin state

changes of the heme iron which can be recorded spectroscopically (Jefcoate, 1978). Yeast

microsomes harboring CYP71E7 produced a substrate binding spectrum with a trough at 415

nm upon addition of ileox (Fig. 4A) which reflects binding of ileox to the CYP71E7 active

site. To quantify the affinity of CYP71E7 for ileox, increasing concentrations of ileox (0.2 to

11.0 μM) were added to the yeast microsomes harboring CYP71E7. From this a KS of 0.9 ±

0.2 μM was calculated. The amplitude of the substrate binding spectrum reached maximum at

3 μM ileox (Fig. 4A). This demonstrated that ileox is a high affinity ligand of CYP71E7.

Cytochromes P450 are known to produce type II spectra upon binding of nitrogen containing

inhibitors such as n-octylamine. This spectral change is indicative of proximity of the

inhibitor amine to the active site heme iron (Jefcoate, 1978). Saturation of CYP71E7 with n-

octylamine (100 μM) resulted in the formation of a typical type IIb binding spectrum with a

trough at 410 nm and a peak at 435 nm [Fig. 4B(1)]. A new baseline was recorded by

addition of 100 μM n-octylamine to the CYP71E7 microsomes in the reference cuvette [Fig.

4B(2)]. To measure the ability of ileox to displace n-octylamine from the active site of

CYP71E7, increasing concentrations of ileox were added to the n-octylamine saturated

CYP71E7 microsome solution. Addition of ileox produced a reverse type IIb spectrum with a

peak at 410 nm and a trough at 430 nm [Fig. 4B (3, 4, 5)]. The amplitude of the reverse type

IIb spectrum reached maximum upon addition of 30 μM ileox. Addition of valox and pheox

to the yeast microsomes harboring CYP71E7 likewise resulted in substrate binding spectra

with a trough at 415 nm (results not shown), although higher substrate concentrations were

required to produce the spectral shift compared to ileox. Only upon addition of >30 μM

substrate did tyrox result in a weak trough at 415 nm (results not shown). The broad substrate

affinity of CYP71E7 is therefore reflected in the ability to bind both aliphatic and aromatic

oximes. The ability of ileox to efficiently replace n-octylamine shows that ileox is a high

affinity ligand to CYP71E7 and that n-octylamine is a competitive inhibitor that also is able

to bind to the active site of CYP71E7.



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CYP71E7 and CYP79D1 are co-expressed in M. esculenta leaf petioles

In tube in situ PCR was used to determine the cellular location of CYP71E7 transcripts in

M. esculenta. The analyses were performed on tissue sections from the petiole and leaf blade

of the nearly unfolded leaf and of the leaf blade of the first fully unfolded leaf using two

month old M. esculenta plants, and with primers that enabled detection of both CYP71E7

paralogs. The young leaf stages were selected because they contain the highest concentration

of cyanogenic glucosides (Jørgensen et al., 2005a) and hence were expected to exhibit high

expression levels of the mRNAs that encode the biosynthetic enzymes. Strong expression of

CYP71E7 in the petiole was found in outer cortex cells and in the cell layer corresponding to

the endodermis, around phloem cells and laticifers as well as in cells surrounding the xylem

as visualized by alkaline phosphatase staining (Fig. 5 CYP71E7). The same expression

pattern is seen for CYP79D1 transcripts in sections from the same petiole (Fig. 5 CYP79D1).

Alkaline phosphatase staining could not be detected in the absence of CYP71E7 or CYP79D1

primers in the reverse transciptase reaction (negative control; Fig. 5 Control). In a separate set

of experiments (Fig. 5), the in situ co-localization of the CYP71E7 and CYP79D1 transcripts

in the petiole were visualized using FITC (Fluorescein isothiocyanate) labeled anti-bodies

against digoxigenin (DIG) incorporated during the PCR reactions. As in the experiment with

alkaline phosphatase, the outer cortex cells, endodermis cells, the cells surrounding the

phloem and laticifers, and cells surrounding the xylem showed strong labeling. Negative

control sections showed no background staining.

In the corresponding leaf blade of the first nearly unfolded leaf, expression of CYP71E7 and

CYP79D1 is observed in most cells in the cortex and in the epidermis (fig.6 A & B,

respectively). In fully unfolded leaves, strong expression of CYP79D1 is observed in the

cortex cell layer just beside the epidermis and in specific cells in the vascular tissue (fig.6 C

& D). Especially in the leaf section red fluorescence is observed due to auto-fluorescence

from the chloroplast. In a previous study, CYP79D1 and CYP79D2 were shown to have

similar expression patterns (Jørgensen et al. 2005a).

The co-expression of CYP71E7 and CYP79D1 further substantiates that CYP71E7 is the

oxime-metabolizing enzyme in cyanogenic glucoside biosynthesis in M. esculenta.

DISCUSSION

A P450 enzyme, designated CYP71E7, catalyzing the conversion of isoleucine- and

valine-derived oximes into the corresponding cyanohydrins in the biosynthesis of cyanogenic



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glucosides in M. esculenta was identified based on amino acid sequence homology to

CYP71E1, functional expression in yeast, substrate binding spectra and transcriptional co-

localization with CYP79D1 and CYP79D2, the genes encoding the two enzymes that catalyze

the initial steps in cyanogenic glucoside synthesis in M. esculenta. The cyanohydrins (2-

hydroxy-2-methylbutyronitrile and acetone cyanohydrin) produced by CYP71E7 are labile

and decompose into volatile ketones (2-butanone and acetone) and HCN. To enable

identification and quantification, the ketones were detected as 2,4-dinitrophenylhydrazone

derivatives by LC-MS. This method enabled detection of pmolar amounts of ketones formed

in the CYP71E7 containing reaction, and is thus more sensitive than the previously used

colorimetric cyanide assay which requires the presence of nmolar amounts (Halkier and

Møller, 1991).

The oxime-metabolizing P450s in cyanogenic glucoside biosynthesis are highly efficient

enzymes

Ileox is a high affinity substrate for CYP71E7 with KS ~0.9 ± 0.2 μM which is reflected in

the conversion of ileox into the corresponding cyanohydrin with a Km of 21 ± 2 μM and a

turnover rate of 17 ± 1 min-1. A similar conversion rate was observed for valox with a

turnover of 21 ± 2 min-1. The higher turnover of valox as compared to ileox is in agreement

with a 97:3 ratio of linamarin and lotaustralin in cassava (Lykkesfeldt and Møller, 1994). The

KS , Km and turnover rates are in the same order of magnitude as those observed for the other

known plant oxime metabolizing cytochrome P450 involved in biosynthesis of cyanogenic

glucosides (CYP71E1) and as those for the structurally related glucosinolates (CYP83A1 and

CYP83B1) (Møller and Conn, 1979; Koch et al., 1992; Bak et al., 2001, Bak and Feyereisen

2001, Hansen et al., 2001, Naur et al., 2003). In contrast, the Km values for CYP79D1 towards

isoleucine and valine are several magnitudes higher, namely 1.3 mM and 2.2 mM,

respectively (Andersen et al., 2000). The Km values for Lotus japonicus (bird’s foot trefoil)

CYP79D3 and CYP79D4 towards isoleucine and valine are also in the 1-3 mM range

(Forslund et al., 2004). Earlier experiments using microsomes prepared from M. esculenta,

Linum usitatissimum (flax) and Triglochin maritima (seaside arrow grass) likewise

demonstrated a KM in the mM range towards the amino acid and a KM in the μM range for the

oxime (summarized in Koch et al., 1992). However, in S. bicolor KM values in the μM range

were obtained for both tyrosine and tyrox (Møller and Conn, 1979). It has been argued that an

enzyme that catalyzes the first committed step in biosynthesis of a cyanogenic glucoside

should have an elevated Km towards its substrate amino acid, to avoid depleting the free



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amino acid pool in the plant (Andersen et al., 2000). In contrast, the oxime-metabolizing

enzyme is expected to have a low Km because the oximes must be metabolized efficiently in

order to prevent release of free oximes which are known to be toxic to the plant

(Grootwassink et al., 1990; Bak et al., 1999; Hemm et al., 2003; Bak et al., 2001; Bak and

Feyereisen 2001; Morant et al., 2007, Morant et al., 2010). The combination of the

biosynthetic enzymes organized in a metabolon and a highly efficient oxime-metabolizing

enzyme explains why no oxime intermediates, or derivatives therof, are detected in

cyanogenic plants (Bak et al., 1999; Tattersall et al., 2001; Kristensen et al., 2005).

Alternatively, a functioning dependent dissociation of the metabolon might result in oxime

production and serve to combat fungal attack (Møller, 2010).

In S. bicolor, the CYP71E1 catalyzed oxime to cyanohydrin conversion proceeds via an

initial dehydration of the oxime to produce a nitrile which is then C-hydroxylated to yield the

cyanohydrin (Fig 1; Kahn et al., 1997; Bak et al., 1998). The CYP71E1 catalyzed

dehydration is an unusual cytochrome P450 reaction and may represent a relict reaction type

reflecting that P450s are ancient enzymes that originally were operating in an anaerobic

environment (Nebert and Feyereisen, 1994). P450 catalyzed dehydrations of oximes are also

known from human CYP3A4 (Boucher et al., 1994), where the reaction catalyzed by

CYP3A4 has been shown to involve direct binding of the nitrogen atom of the oxime

function to the heme iron of the P450 (Hart-Davis et al., 1998). The iron needs to be in its

reduced state (FeII) to bind the substrate, and this may explain why this reaction is NADPH

dependent. M. esculenta CYP71E7 and S. bicolor CYP71E1 catalyze the same reaction and

we propose that CYP71E7 is also bi-functional and catalyzes an initial dehydration of the

aldoxime followed by a C-hydroxylation to yield the cyanohydrin which, by glucosylation, is

converted into a cyanogenic glucoside.

CYP71E7 has broad substrate specificity

Cyanogenic glucosides are produced from the protein amino acids isoleucine, valine,

leucine, tyrosine and phenylalanine and from the non-protein amino acid cyclopentenyl

glycine.

In addition to the aliphatic oxime intermediates in lotaustralin and linamarin biosynthesis,

CYP71E7 catalyzes the conversion of tyrox and pheox, albeit with lower efficiency as

compared to valox and ileox. This property is in agreement with results of previous studies

using M. esculenta microsomes that indicated that these had the ability to metabolize

aliphatic as well as aromatic oximes into the corresponding cyanohydrins (Koch et al., 1992).



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Low substrate specificity at the oxime level was also observed using microsomes from L.

japonicus (Forslund et al., 2004). Studies in transgenic plants likewise support low substrate

specificity at the oxime level (Morant et al., 2007). These studies all provided evidence that

the substrate specificity for cyanogenic glucosides and glucosinolate biosynthesis is

determined at the parent amino acid level and that in both pathways the post oxime enzymes

have low specificity for the side chain of the substrate. The lack of need for high substrate

specificity of the post oxime enzymes can be explained by assembly of the biosynthetic

enzymes into a metabolon and by absence of other oximes (Jørgensen et al., 2005; Kristensen

et al., 2005; Nielsen et al., 2008). The organization of the biosynthetic pathway within a

metabolon imposes an evolutionary constraint for narrow substrate specificity only on the

first enzyme in the pathway.

Evolutionary origin of the two CYP71E7 paralogs

M. esculenta CYP79D1 and CYP79D2 catalyze the first committed steps in lotaustralin

and linamarin biosynthesis and exhibit the same catalytic parameters (Andersen et al., 2000).

CYP79D1 and CYP79D2 possess ~85% amino acid sequence identity, and CYP79D1 and

CYP79D2 show identical expression patterns based on in tube in situ PCR (Jørgensen et al.,

2005a). M. esculenta is an allopolyploid, and the relatively low sequence identity between

CYP79D1 and CYP79D2 most likely reflects that they are homeologs that originate from

separate ancestral diploid parental genomes, rather than via a gene or genome duplication

event. In support of this CYP79D1 and CYP79D2 are located on separate scaffolds. In

contrast, the two CYP71E7 paralogs are located on scaffold 02340 and are 90% identical and

94% similar on the amino acid level. In addition, both genes are located within a 12 kbp-

region and in close proximity to CYP79D2 which is on the same scaffold. This indicates that

the two paralogs have arisen by a recent gene duplication event and thus originate from the

same ancestral diploid parental genotype. Hence, it might be expected that a homeolog

CYP71E7 could exist in the M. esculenta genome originating from a parental genome

different to the one harboring the two CYP71E7 paralogs. However we have not been able to

identify such a homeolog in the current 1.1 version of the cassava genome .

The L. japonicus genome also contains two CYP79D paralogs. The sequence identity

between these paralogs is ~95% at the amino acid level and they catalyze the same enzyme

reaction. However, their promoters have diverged and their expression patterns differ

(Forslund et al., 2004). This indicates that the two CYP79D paralogs in L. japonicus have

originated from a gene or whole genome duplication event and that subsequent



15

subfunctionalization has led to differences in expression patterns while catalysis is

maintained. The fact that L. japonicus is a paleopolyploid where the most recent

autopolyploidy event happened more than 40 million years ago preceding the speciation of L.

japonicus and Medicago truncatula (barrel clover; Cannon et al., 2006) is in support of this

assumption.

CYP71E7 and CYP79D1 transcripts co-localize in M. esculenta

CYP71E7 mRNA co-localizes with CYP79D1 mRNA in M. esculenta petioles and leaves.

This substantiates that CYP71E7 is the oxime-metabolizing enzyme in cyanogenic glucoside

biosynthesis in M. esculenta. The cyanogenic glucoside degrading β-glucosidase is located in

the cell walls and in the laticifers in M. esculenta (Elias et al., 1997), and thus expression of

the biosynthetic genes in outer cortex, in endodermis and in cells adjacent to the laticifers

ensures that the cyanogenic glucosides and their bio-activators are in close proximity, yet

separated into different cell types or subcellular compartments. The observed expression of

CYP71E7 and CYP79D1 in cells surrounding phloem could indicate that the cyanogenic

glucosides synthesized in these cells are destined for transport.

Cyanogenic glucosides are synthesized via an evolutionarily conserved pathway in

monocotyledons and eudicotyledons

S. bicolor is monocotyledonous and produces the aromatic cyanogenic glucoside dhurrin

while M. esculenta is a eudicotyledon and synthesizes aliphatic cyanogenic glucosides. The

identification of CYP71E7 and CYP71E1 as functional ortholog confirms that cyanogenic

glucosides are synthesized via an evolutionarily conserved biosynthetic pathway (Jones et al.,

2000, Bak et al., 2006). This will aid identification of CYP71E1 orthologs from other

eudicotyledonous cyanogenic plants such as the model legume L. japonicus and crop plants

like Trifolium repens (white clover), Prunus dulcis (almond), Prunus avium (sweet cherry),

Phaseolus lunatus (lima bean) and Phaseolus vulgaris (kidney bean). These plants all

produce aliphatic cyanogenic glucosides and CYP71E7 would be expected to serve as an

ideal probe for isolation of paralogs in those plants.

Identification and characterization of the enzymes involved in biosynthesis, transport and

degradation of cyanogenic glucosides in M. esculenta will provide the necessary molecular

tools to enable production of transgenic M. esculenta lines in which the cyanogenic glucoside

content of various tissues is optimized to achieve pest resistance while retaining acyanogenic



16

tubers. Such acyanogenic M. esculenta tubers will provide a healthier diet for millions of

people especially in developing countries.



17

MATERIALS AND METHODS

Isolation and expression of CYP71E7 in Saccharomyces cerevisiae

The M. esculenta CYP71E7 cDNA (accession no. AY217351) was PCR amplified using a

plasmid cDNA library made from shoot tips of M. esculenta plants of MCol22 (Andersen et

al., 2000) as a template and primers 5’-

ggtggtggtggatccATGTCCGTAGCCATTCTAACCTCACTG-3’ and 5’-

ccaccaccagaattcTCAGTCCCATTTGTGCTTTTTAGGAAT-3’ harboring EcoRI and BamHI

restriction sites (underlined). PCR reactions (total volume: 50 μL) were carried out in 10 mM

Tris-HCl (pH 8.3), 50 mM KCl and 2 mM MgSO4 containing 2.5 U Pwo DNA polymerase

(Roche Diagnostics A/S, Denmark), 50 μM dATP, 50 μM dCTP, 50 μM dGTP, 50 μM dTTP

and 10 pmoles of each of the primers listed above. Thermal cycling parameters were: 94ºC

for 2 min followed by 30 cycles of (94ºC for 30 s, 65ºC for 60 s and 72ºC for 90 s) and a final

72ºC for 5 min. The purified PCR product was ligated into the EcoRI and BamHI sites of

pYeDP60 (Pompon et al., 1996) to yield pYeCYP71E7. The authenticity of the insert was

verified by DNA sequencing. The unknown nucleotide at position 189 from the ATG start

codon (Zhang et al., 2003) was hereby shown to be a T. pYeCYP71E7 was transformed into

S. cerevisiae WAT11, expression of CYP71E7 was induced by galactose addition, and

microsomes were obtained as previously described (Pompon et al., 1996). Recombinant

CYP71E7 was quantified by CO difference spectroscopy (Omura and Sato, 1964).

Catalytic activity of CYP71E7

Yeast microsomes harboring recombinant CYP71E7 were assayed for the ability to

convert ileox and valox into the corresponding cyanohydrins using assay mixtures (total

volume: 100 μL) containing: ~0.20 μM CYP71E7, 10 mM Tricine (pH 7.9), 1 mM NADPH

and 0-100 μM 2-methylbutanal oxime or 2-methylpropanal oxime. The ileox and valox

administered to CYP71E7 are mixtures of approximately 70% (E)-isomers and 30% (Z)-

isomers as determined by 1H NMR. Assay mixtures without NADPH or containing

microsomes from S. cerevisiae WAT11 transformed with an empty pYeDP60 vector served

as negative controls. Incubation (30 min, 28ºC, 300 rpm) was performed in glass vials (1.5

mL) fitted with a gas tight silicone stopper and a centre well composed of a plastic pipette tip

melted at the end to form a closed cone inside the glass vial. Enzyme reaction was stopped by

direct injection of NaOH (10 μL, 2.5 N). To trap volatile ketones, an acidified 2,4-di-



18

nitrophenylhydrazine (DNPH) solution [50 μL, 6.66 mg DNPH dissolved in 1.25 ml HCl,

3.125 ml H2O and 0.625 mL acetonitrile (Zwiener et al., 2002), thrice extracted with n-

pentane to remove aldehyde and ketone contaminants] was injected into the center well and

incubation continued (2h, 50ºC, 300 rpm) to trap the ketones as hydrazones in the center well.

For LC-MS analysis, hydrazones were extracted into n-pentane (600 μL) and the pellet

obtained after removal of the n-pentane dissolved in methanol (85%, 60 μL). The amount of

hydrazone present was determined from the extracted ion chromatogram (EIC) based on the

area under the appropriate signals compared to those generated by known standards (100 μL

of 0, 1, 5, 10, 25, 50 and 100 μM solutions of acetone or 2-butanone) applied to the

incubation vials and trapped by diffusion into the DNPH containing center well. For

calculation of turnover numbers, assays were performed in four replicates as described above,

except that 0.026 μM CYP71E7 was applied with 100 μM substrate in the presence or

absence of NADPH, and incubation reduced to 10 min. Turnover numbers were calculated

based on the difference of oxime conversion in the presence and absence of NADPH.

Standard deviations were calculated as the difference between the average turnover in the

presence and absence of NADPH. The KM value was calculated based on 10 different Ileox

concentrations ranging from 1 to 100 µM.

Assays to determine turnovers for tyrox and pheox were carried out as described above,

except that 0.26 μM CYP71E7 was applied, incubation was reduced to 10 min, and

termination of the reaction by NaOH (10 μL 2.5 N NaOH, 10 min, 28oC) was followed by

direct injection of DNPH solution (80 μL) into the assay mixture. After incubation (50ºC, 1 h,

300 rpm), hydrazone products formed were extracted as above. Quantification of tyrox and

pheox derived cyanohydrins was based on injection of standards (100 μL of 0, 1, 5, 10, 25, 50

and 100 μM solutions of p-hydroxymandelonitrile or mandelonitrile dissociated by NaOH

addition and reacted with DNPH as described above). Authentic standards were produced by

reacting p-hydroxybenzaldehyde and benzaldehyde (100 μL 100 μM) directly with acidified

DNPH.

LC-MS analysis

LC-MS was performed using a HP1100 HPLC (Agilent Technologies, Germany) coupled

to a Bruker HCT-Ultra ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). An

XTerra MS C18 column (Waters, Milford, MA; 3.5 μM, 2.1 × 100mm) was used at a flow



19

rate of 0.2 mL min-1. The mobile phases were: A, 2% (v/v) formic acid in water; B, methanol.

The gradient program was: 0 to 2 min, isocratic 65% B; 2 to 10min, linear gradient 65 to

100% B; 10 to 12 min, isocratic 100% B, 12.1 to 18 min, isocratic 65% B. The spectrometer

was run in positive APCI mode using a vaporizer temperature of 400°.

Substrate binding monitored by optical difference spectroscopy

All spectra were obtained using a Perkin Elmer spectrometer (Lambda 800) and

microsomes harboring ~0.24 μM CYP71E7 resuspended in TEG buffer [50 mM Tris HCl

(pH 7.5), 1 mM EDTA, 30% glycerol, total volume: 500 μL] in sample and reference

cuvettes (1 cm light path). To monitor spectral changes upon substrate binding, a base line

was recorded using CYP71E7 expressing microsomes alone in both cuvettes. Binding spectra

were recorded following addition of ileox (0.5, 1 and 3 µM) or n-octylamine (100 μM) to the

sample cuvette. A KS for ileox was calculated using SigmaPlot (Systat Software Inc., CA)

from the increase in magnitude between trough and peak in the binding spectra recorded

following addition of increasing concentrations of ileox (9 concentrations between 0.2 and 11

μM). Displacement of the inhibitor n-octylamine from the CYP71E7 active site by ileox was

measured as the spectral shifts observed upon addition of increasing amounts of ileox (1, 10

and 30 μM) to CYP71E7 harboring microsomes in the presence of n-octylamine (100 μM)

after recording a new base line upon addition of n-octylamine (100 μM) to the reference

cuvette.

In tube in situ PCR on tissue sections of M. esculenta leaf petioles

In tube in situ PCR was carried out on sections of nearly unfolded leaves, fully unfolded

leaves and on petioles from the first fully unfolded leaf from two months old M. esculenta

plants of MCol22 as previously described (Jørgensen et al., 2005a) using primers 5’-

GGATGGTGCATATCCAAACC-3’ and 5’-GCCTTGACATGATCCTTGGT-3’ for

CYP71E7 and 5’-CTTCTTCAGGATTTCTGGTTGATT-3’ and 5’-

AATTTGTGCTTGATGCAAATAAGA-3’ for CYP79D1. The specificity of the primers was

verified by DNA sequencing of the PCR products. Prior to sectioning, all tissues were fixed

in FAA (2% formaldehyde; 5% acetic acid; 63% ethanol in PBS) for 5 h at 4ºC and then

washed 3 times with washing buffer (60% ethanol, 5% acetic acid in PBS). The tissue was

embedded in 5% agarose in PBS to enable sectioning into 80µm tissue sections on a Leica

vibratome. The sections were treated overnight with 30 µl RNase free DNAse (2u/µl) and 30

µl RNasin (0.8mu/µl) at 37ºC, and subsequently washed twice with H2O. To partly release



20

the co-agulation of the fixative, the sections were incubated in 30 µl Pepsin (2mg /l, 0.1M

HCl) for 12 min. prior to the reverse transcriptase reaction, the sections were washed twice in

H2O. The mRNA was translated to cDNA using the specific reverse primers for CYP71E7

and CYP79D1, respectively, using the protocol from Sensiscript® (Qiagen 1017746). Before

the cDNA was amplified, the reverse transcriptase solution was removed and the PCR

reagents were added. DIG labeled dUTP was incorporated as basis for visualization. The

expression was subsequently visualized using either FITC or alkaline phosphatase labeled

antibodies recognizing DIG. The sections were mounted on glass slides and examined using a

Leica DM microscope. The sections stained with the FITC labeled antibodies recognizing

DIG were examined using a FI/RH filter cube from Leica.



21

ACKNOWLEDGEMENTS

We are very grateful to Dr. René Feyereisen for helpful discussions on the detection of

CYP71E7 products. We thank Dr. Nanna Bjarnholt for helpful discussions and Mr. Steen

Malmmose is thanked for maintaining the M. esculenta plants. Susanne Bidstrup and Ziff

Hansen are thanked for their excellent technical assistance. Dr. Claus Thorn Ekstrøm is

thanked for help on statistical calculations. Emma O’Callahan is thanked for proof reading

the manuscript.



22

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27

FIGURE LEGENDS

Figure 1. Biosynthesis of the isoleucine- and valine-derived cyanogenic glucosides

lotaustralin and linamarin in M. esculenta with emphasis on the CYP71E7 catalyzed

reaction. The conversion of isoleucine and valine 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-glucosyl transferase (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

extracted ion chromatograms (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); 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: (1) Baseline recorded with CYP71E7 harboring microsomes in the absence

of substrate in both sample and reference cuvettes; (2,3,4) Spectra after addition of ileox (0.5,

1 and 3 μM, respectively) to sample cuvette. B: (1) Spectrum of CYP71E7 harboring

microsomes saturated with the cytochrome P450 inhibitor n-octylamine (100 μM); (2)

Baseline recorded upon addition of equal amounts of n-octylamine (100 μM) to CYP71E7



28

harboring microsomes in both sample and reference cuvette; (3,4,5) Displacement of n-

octylamine by addition of ileox (1,10 and 30 μM, respectively) to 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 RT-PCR analysis of 80 µm transverse sections. c:

cortex, e:endodermis, l: laticifers and x: xylem. Bar: 100 µm. The expression of the

transcripts has been visualized using either alkaline phosphatase labeled (black staining) or

FITC labeled (bright light green staining) antibodies recognizing DIG.

Figure 6: Cellular localization of the expression of CYP79D1 and CYP71E7 in two leaf

stages, A nearly unfolded leaf and of the first fully unfolded leaf, using in tube in situ RT-

PCR analysis of 80 µm transverse sections. A and B localization of respectively CYP79D1

and CYP71E7 expression in a nearly unfolded upper leaf. C and D localization of CYP79D1

expression in an unfolded leaf. e: epidermis, cp: pallisade tissue, v: vascular tissue. Bar

100µm. The expression of the transcripts has been visualized using FITC labeled (bright light

green staining) antibodies recognizing DIG.



Figure 1. Biosynthesis of the isoleucine- and valine-derived cyanogenic glucosides lotaustralin and linamarin in M. esculenta with emphasis on the CYP71E7 catalyzed reaction. The conversion of isoleucine and valine 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-glucosyl transferase (UGT) provides lotaustralin and linamarin.

COOH

NH2

CH3

R

N

CH3

R

OH

CH3

RCN

R

OH

CH3

CN

R

O

CH3

CN

Glu

R=H: L-valineR=CH3: L-isoleucine

CYP79D1

CYP79D2

NADPH

O2 + NADPH

NADP+

R=H: acetone cyanohydrinR=CH3: 2-hydroxy-2-methylbutyronitrile

R=H: linamarinR=CH3: lotaustralin

CYP71E7

UGT functional homologue

H2O + NADP+

R=H: (Z)-2-methylpropanal oxime (valox)R=CH3: (Z)-2-methylbutanal oxime (ilox)



390 420 450 480 500

-0,02

0,00

0,02

0,04

nm

A

Figure 2. Carbon monoxide difference spectrum of yeast microsomes harbouring CYP71E7. The Fe2+.CO versus Fe2+

difference spectrum was recorded on the microsomal fraction of yeast expressing CYP71E7.

Abs

orba

nce

Wavelength (nm)



O

CH3

R

+NO2

NO2

NHNH2

NO2

NO2

NHN

CH3

R

pH<1

50oC

R=H: acetone

R=CH3: 2-butanone

2,4-dinitrophenylhydrazine (DNPH)

R=H: acetone 2,4-dinitrophenylhydrazone

R=CH3: 2-butanone 2,4-dinitrophenylhydrazone

OH

CH3

CN

R

R=H: acetone cyanohydrin

R=CH3: 2-hydroxy-2-methylbutyronitrile

CN- + H2O

(g)

(aq)

(aq)

O

CH3

R

R=H: acetone

R=CH3: 2-butanone

(g)

(aq)

OH-

+ H2O

A

EIC 253 EIC 239

5.5 6.5 7.59.0 10.0 11.00.0

0.5

1.0

1.5

C

B

Inte

nsit

y x

10-5

Retention time (min)

Figure 3. Analysis of CYP71E1 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 extracted ion chromatograms (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); CYP71E7 containing yeast microsomes, 100 μM oxime + 1 mM NADPH (dark green, dotted).



2

-2

0

8

0

-4

4

370 400 420 450 380 420 460

Abs

orba

nce

x 10

3

nm nm

A

1

1

2 2

3

3

4

4

5

BA

Wavelength (nm)

Figure 4. Substrate binding properties of CYP71E7 as analyzed by optical difference spectroscopy. A: (1) Baseline recorded with CYP71E7 harbouring microsomes in the absence of substrate in both sample and reference cuvettes; (2,3,4) Spectra after addition of ilox (0.5, 1 and 3 μM, respectively) to sample cuvette. B: (1) Spectrum of CYP71E7 harbouring microsomes saturated with the cytochrome P450 inhibitor n-octylamine (100 μM); (2) Baseline recorded upon addition of equal amounts of n-octylamine (100 μM) to CYP71E7 harbouring microsomes in both sample and reference cuvette; (3,4,5) Displacement of n-octylamine by addition of ilox (1,10 and 30 μM, respectively) to sample cuvette.



Figure 5: Cellular localisation of the expression of CYP79D1and CYP71E7 in the petiole of the first unfolded leaf using in tube in situ RT-PCR analysis of 80 µm transverse sections. c: cortex, e:endodermis, l: laticifers and x: xylem. Bar: 100 µm. The expression of the transcripts has been visualised using either alkaline phosphatase labelled (black staining) or FITC labelled (bright light green staining) antibodies recognising DIG.

l

c

l

c

x

c

l

x

c

x

l

c

l

x

c

x

l

c

xl

c

x

l

c

x

l

Expression of mRNA monitored by

Alkaline Phosphatase

Light microscopy FITC control

CYP79D1 expression monitored by FITC

CYP71E7 expression monitored by FITC

CYP79D1 Control CYP71E7

e e e

e e

e e

e e



A

DC

Be

e

cp

cp

cp

cp

e

v

v

Figure 6: Cellular localization of the expression of CYP79D1 and CYP71E7 in two leaf stages, a nearly unfolded leaf and of the first fully unfolded leaf, using in tube in situ RT-PCR analysis of 80 µm transverse sections. A and B localization of respectively CYP79D1and CYP71E7 expression in a nearly unfolded upper leaf. C and D localization of CYP79D1 expression in un unfolded leaf. e: epidermis, cp: pallisade tissue, v: vascular tissue. Bar 100µm. The expression of the transcripts has been visualised using FITC labelled (bright light green staining) antibodies recognising DIG.



Ilox Valox Tyrox Pheox

Turnover (min-1) 17.1 ± 1.0 21.2 ± 2.2 8.1 ± 0.3 1.3 ± 0.2

Km (µM) 21 ± 11 n.d. n.d. n.d.

Table 1

Table 1. Kinetic parameters of CYP71E7 catalyzed oxime to cyanohydrin conversions.



RUNNING HEAD: CYP71E7: The oxime-metabolizing P450 in ...4 ABSTRACT Cassava (Manihot esculenta...

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Transcript of RUNNING HEAD: CYP71E7: The oxime-metabolizing P450 in ...4 ABSTRACT Cassava (Manihot esculenta...