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A cytochrome P450 from juvenile mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis

10 May 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 634485
TL;DR: The geraniol 8-hydroxylase in Phaedon cochleariae was identified as a cytochrome P450 CYP6BH5, and homology modeling suggested the -OH group of the substrate plays an important role in coordinating the substrates with the enzyme’s catalytic cavity.
Abstract: Juveniles of the leaf beetle Phaedon cochleariae synthesize iridoid via the mevalonate pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated defensive pathway by the {omega}-hydroxylation of geraniol to 8-hydroxygeraniol. Here we identify and characterize the geraniol 8-hydroxylase as a P450 monooxygenase using integrated transcriptomic and proteomic analyses. In the fat body, 73 individual cytochrome P450s were identified. The double stranded RNA (dsRNA)-mediated knock down of CYP6BH5 led to a significant reduction of 8-hydroxygeraniol-glucoside in the hemolymph and, later, of the chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted geraniol to 8-hydroxygeraniol. In addition to geraniol, CYP6BH5 also catalyzes other monoterpenols, such as nerol and citronellol, into the corresponding , {omega}-dihydroxy compounds.nnHighlightsO_LIThe geraniol 8-hydroxylase in Phaedon cochleariae was identified as a cytochrome P450 CYP6BH5.nC_LIO_LIRNA interference emphasized the importance of CYP6BH5 in iridoid biosynthesis.nC_LIO_LIIn vitro enzyme assays showed that recombinant CYP6BH5 is a substrate promiscuous enzyme, converting the {omega}-hydroxylation of geraniol, nerol, citronellol but not linalool.nC_LIO_LIHomology modeling suggested the -OH group of the substrate plays an important role in coordinating the substrates with the enzymes catalytic cavity.nC_LI

Summary (2 min read)

2.2. RNA extraction and cDNA synthesis

  • The cDNA template from the P. cochleariae fat body for 3' rapid amplification of cDNA ends (3' RACE) was synthesized according to the manual from SMARTer RACE 5'/3' Kit (Takara Bio, Inc. Mountain View, CA, USA).
  • Proteins of the membrane fractions were separated by SDS PAGE and stained afterwards with Coomassie blue.
  • The proteomic data were processed with their in-house P. cochleriae transcriptome database (Stock et al., 2013) .
  • Afterwards, Pfam and Blast2Go analysis were used to identify all fat body cytochrome P450s.

2.4. Analysis of the expression pattern in selected tissues

  • The assembled sequences of selected candidates were analyzed by siFi 21 (https://sourceforge.net/projects/sifi21/) for off-target prediction using a threshold value of at least 21 continuous nucleotides.
  • The unique fragments (>400 bp) were used to design dsRNA primers (Table S3 ) and all dsRNA fragments (including the ds eGFP control) were synthesized using the MEGAscript RNAi Kit (Thermo Fisher Scientific, Langenselbold, Germany).
  • After purification, the dsRNA was diluted in 0.9% NaCl solution and adjusted to a concentration of 2 μg/µl.
  • Second instar P. cochleariae larvae were anesthetized on ice prior to injection.
  • Larvae injected with only dsRNA targeting eGFP were used as a control group.

2.6. Collection of hemolymph and the glandular secretion

  • Larval glandular secretion and hemolymph were collected by glass capillaries (i.d.: 0.28 mm, o.d.: 0.78 mm, length: 100 mm; Hirschmann, Eberstadt, Germany) and then transferred to a 200 μl Eppendorf tube.
  • The weight of the glandular secretion and hemolymph were obtained by weighing empty and filled tubes with hemolymph or secretion.
  • Samples were stored at -20 °C until needed.

2.7. Amplification and sub-cloning of the PcC7758 open reading frame (ORF)

  • To produce the recombinant protein, CYP6BH5 was sub-cloned into pcDNA™3.1D/V5-His-TOPO and pFastBac Dual expression vectors, respectively, according to the protocol (Thermo Fisher Scientific, Langenselbold, Germany).
  • To facilitate detection of the recombinant proteins, constructs without a stop codon were sub-cloned in parallel into corresponding expression vectors.
  • A C. populi cytochrome P450 reductase (CPR) was cloned and subjected to operations similar to those mentioned above.

2.8. Expression of the recombinant proteins and microsome isolation

  • The copyright holder for this preprint (which was not this version posted May 10, 2019.
  • After 72 h, cells were harvested for isolation of the microsomal fraction.
  • The microsomal fraction was aliquoted and stored at -80 °C after protein quantification with the Quick Start Bradford Protein Assay Kit (Bio-Rad Laboratories, Munich, Germany).

2.9. SDS PAGE and western blot

  • Microsomal fractions containing CYP6BH5 and CPR, fused with a V5 epitope tag and a His-tag were denatured by incubation at 70 °C for 5 minutes and followed by SDS PAGE separation.
  • Afterwards, membrane proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Trans-Blot ® Turbo™ Mini PVDF Transfer Pack; Bio-Rad) with a Bio-Rad blotting system.
  • The membrane was blocked first in blocking buffer (5% (wt/vol) non-fat dry milk in Tris buffered saline with Tween-20 (TBST) buffer) for 1 h and then incubated overnight with Anti-V5-HRP antibody (1:10,000; Thermo Fisher Scientific, Langenselbold, Germany) in another blocking buffer (0.25 % (wt/vol) non-fat dry milk in TBST buffer) at 4 °C.
  • The membrane was washed three times with TBST buffer prior to incubation (1 min) with enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific, Langenselbold, Germany).
  • The Amersham Hyperfilm ECL X-ray film (GE Healthcare, Boston, USA) was exposed to the PVDF membrane prior to developing the film.

2.10. Enzyme assays

  • Two up-scaled assays for each substrate were pooled.
  • To extract the products, equal volumes of ethyl acetate were added and the supernatants were collected.
  • The solvent of the samples was subsequently removed by a stream of nitrogen gas.

3.1. Presence of 8-OH-geraniol in the fat body of P. cochleariae

  • The aglycone 8-OH-geraniol is the first metabolite that links classical terpenoid biosynthesis to dedicated defense metabolism.
  • The presence of the corresponding aglycone in the fat body has not been validated.
  • Therefore, the presence of 8-OH-geraniol in this tissue was checked first.
  • Moreover, its glucoside could also be detected in the fat body with HPLC-MS (Fig. S2 ).
  • This observations support the hypothesis that the early biosynthetic steps of the iridoid biosynthesis pathway are localized in the fat body (Burse et al., 2007) , which therefore should contain corresponding enzymes including the geraniol oxidase and the glycosyltransferase.

3.5. Homology modeling of CYP6BH5 with the substrate geraniol

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  • Furthermore, the side chain of Val365 supports the correct positioning by hydrophobic interactions with the prenyl moiety.
  • The transoid methyl group has a distance of 3.2 Å and the cisoid methyl group of 3.4 Å to the distal oxygen atom sitting in the middle of the heme center.
  • Therefore, the enzyme will preferentially lead to oxidation of the trans methyl group.

4. Discussion

  • The elucidation of the involved enzymes in the iridoid biosynthesis pathway in both P. cochleariae and C. roseus lays the foundation for producing these pharmacologically valuable monoterpenoid indole alkaloids on an industrial level.
  • Recent, attempts to reconstruct the biosynthetic pathways for nepetalactol, vindoline and strictosidine from the geranyl diphosphate in yeast Saccharomyces cerevisiae have made impressive progress (Billingsley et al., 2017; Brown et al., 2015; Campbell et al., 2016; Qu et al., 2015) .
  • The newly identified CYP6BH5 from P. cochleariae, therefore, may serve as an alternative biocatalyst for overcoming this obstacle, further boosting the yields of monoterpenoid indole alkaloids.

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Figures (6)

Content maybe subject to copyright    Report

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A cytochrome P450 from juvenile mustard leaf beetles hydroxylates geraniol,
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a key step in iridoid biosynthesis
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Nanxia Fu
1
, Zhi-ling Yang
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, Yannick Pauchet
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, Christian Paetz
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, Wolfgang Brandt
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, Wilhelm
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Boland
1*
, Antje Burse
1, 6*
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1
Department of Bioorganic Chemistry, Max Planck Institute for Chemical Ecology, Hans Knöll
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Str. 8, 07745 Jena, Germany
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Research Group Sequestration and Detoxification in Insects, Max Planck Institute for Chemical
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Ecology, Hans Knöll Str. 8, 07745 Jena, Germany
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3
Department of Entomology, Max Planck Institute for Chemical Ecology, Hans Knöll Str. 8,
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07745 Jena, Germany
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Research Group Mass Spectrometry/ Proteomics, Max Planck Institute for Chemical Ecology,
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Hans Knöll Str. 8, 07745 Jena, Germany
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Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3,
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06120 Halle (Saale), Germany
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Department of Medical Technology and Biotechnology, Ernst Abbe Hochschule Jena, Carl
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Zeiss Promenade 2, 07745 Jena, Germany
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*Corresponding authors:
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certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 10, 2019. ; https://doi.org/10.1101/634485doi: bioRxiv preprint

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Phone: ++49(0) 3641-571265, Fax: ++49(0) 3641-571202, E-mail: aburse@ice.mpg.de
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Phone: ++49(0) 3641-571200, Fax: ++49(0) 3641-571202, E-mail: Boland@ice.mpg.de
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Highlights
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The geraniol 8-hydroxylase in Phaedon cochleariae was identified as a cytochrome P450
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CYP6BH5.
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RNA interference emphasized the importance of CYP6BH5 in iridoid biosynthesis.
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In vitro enzyme assays showed that recombinant CYP6BH5 is a substrate promiscuous
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enzyme, converting the ω-hydroxylation of geraniol, nerol, citronellol but not linalool.
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Homology modeling suggested the -OH group of the substrate plays an important role in
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coordinating the substrates with the enzyme’s catalytic cavity.
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Key words: Chrysomelidae, chemical defense, iridoid, P450 monooxygenase, fat body, Phaedon
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cochleariae
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Abstract
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Juveniles of the leaf beetle Phaedon cochleariae synthesize iridoid via the mevalonate
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pathway to repel predators. The normal terpenoid biosynthesis is integrated into the dedicated
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defensive pathway by the ω-hydroxylation of geraniol to 8-hydroxygeraniol. Here we identify
41
and characterize the geraniol 8-hydroxylase as a P450 monooxygenase using integrated
42
transcriptomic and proteomic analyses. In the fat body, 73 individual cytochrome P450s were
43
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 10, 2019. ; https://doi.org/10.1101/634485doi: bioRxiv preprint

3
identified. The double stranded RNA (dsRNA)-mediated knock down of CYP6BH5 led to a
44
significant reduction of 8-hydroxygeraniol-glucoside in the hemolymph and, later, of the
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chrysomelidial in the defensive secretion. Heterologously expressed CYP6BH5 converted
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geraniol to 8-hydroxygeraniol. In addition to geraniol, CYP6BH5 also catalyzes other
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monoterpenols, such as nerol and citronellol, into the corresponding α, ω-dihydroxy compounds.
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1. Introduction
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Iridoids comprise a large family of biologically active molecules that have so far been found
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in plants and insects. Structurally, they are known as cis-fused cyclopentan-[c]-pyrans with a
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hydroxyl (iridoid aglucones) or glucosyloxy group (iridoid glucosides) at C-1 position of the
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pyran ring. Besides their genuine defensive function against herbivores or insect predators,
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extensive studies have revealed that iridoids are also pharmaceutically valuable for the
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development of novel drugs and therapeutic strategies against diverse conditions including
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inflammation and cancers (Boros and Stermitz, 1991; Ghisalberti, 1998; Laurent et al., 2005;
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Rosa et al., 2008; Yamane et al., 2010). However, the often low yield of iridoids from natural
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resources has greatly hampered their therapeutic application (Miettinen et al., 2014). Maximizing
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the production of iridoids to an industrial scale using metabolic engineering offers a promising
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solution to their current scarcity (Alagna et al., 2016; Brown et al., 2015). To facilitate the
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optimization of the metabolic engineering, the identification of the genes and enzymes required
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for iridoid biosynthesis is essential.
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Madagascar periwinkle, Catharanthus roseus, is a well-characterized iridoid-producing
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medicinal plant. Recently, all genes and enzymes involved in the iridoid biosynthetic pathway
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were completely elucidated in C. roseus (Krithika et al., 2015; Larsen et al., 2017; Miettinen et
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certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 10, 2019. ; https://doi.org/10.1101/634485doi: bioRxiv preprint

4
al., 2014). Although iridoids are typically encountered in the plant kingdom, these secondary
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metabolites have also been identified in insects (Cavill et al., 1984; Oldham et al., 1996; Smith et
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al., 1979). In fact, the name iridoid is a generic term derived from iridomyrmecin, iridolactone
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and iridodial, components of defensive secretions identified from species of the ant genus
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Iridomyrmex (Cavill et al., 1984). However, in insects, the iridoid pathway has been
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characterized in only a few species, and in Iridomyrmex, the pathway is even not yet fully
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resolved. The best-investigated insect species so far belong to the family of leaf beetles
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(Chrysomelidae). In particular, juveniles of Chrysomelina beetles, such as the mustard leaf beetle,
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Phaedon cochleariae, have evolved specialized pair-wise exocrine glands (composed of a
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reservoir with adhering glandular cells) to fend off predators; these glands, located on the
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beetle’s dorsal segment, are able to release defensive droplets containing iridoids. As shown in
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Figure 1, the iridoid de novo biosynthesis in P. cochleariae larvae starts with the formation of the
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early stage precursor 8-hydroxygeraniol glucoside (8-OH-Ger-Glc) in the fat body, which is
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transported via the hemolymph into the glandular reservoir (Burse et al., 2009, 2007; Frick et al.,
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2013; Strauss et al., 2013). In the glandular reservoir, 8-OH-Ger-Glc is subject to sequential
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hydrolysis, oxidation and cyclization to form the ultimate product, chrysomelidial (Bodemann et
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al., 2012; Rahfeld et al., 2015, 2014; Strauss et al., 2013). Although most of the glandular
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enzymes involved in the later steps of the pathway are well understood, to date only a few
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enzymes from the early biosynthetic steps have been functionally characterized (Burse et al.,
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2009; Frick et al., 2013; Kunert et al., 2013; Rahfeld et al., 2015; Strauss et al., 2013). Similar to
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other monoterpernoids, iridoid biosynthesis in P. cochleariae is initiated from geranyl
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pyrophosphate (GPP), a compound that is synthesized from the mevalonate pathway (Frick et al.,
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certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 10, 2019. ; https://doi.org/10.1101/634485doi: bioRxiv preprint

5
2013; Snyder and Qi, 2013). GPP is supposed to be further metabolized by a series of enzymes
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to generate the shuttling glucoside 8-OH-Ger-Glc.
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Stable isotope labeling studies indicated the production of shuttling glucoside 8-OH-Ger-Glc
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in P. cochleariae was likely to proceed as follows (Oldham et al., 1996; Veith et al., 1994). First,
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the phosphate group is first removed from GPP by a presumable phosphatase, yielding geraniol
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(Burse et al., 2007). Afterwards, an oxidase converts geraniol into the diol 8-hydroxygeraniol (8-
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OH-geraniol) (Veith et al., 1994). In the step that follows, a glucosyltransferase catalyzes the
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addition of a sugar moiety to form the desired shuttling glucoside (Kunert et al., 2013). In C.
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roseus, the hydroxylation from geraniol to 8-OH-geraniol is catalyzed by a cytochrome P450
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(Collu et al., 2001). It is hypothesized that the enzymatic system that produces 8-OH-geraniol in
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juvenile P. cochleariae has similar characteristics as in plants. However, the molecular
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characteristics of such an enzyme remain elusive.
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certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 10, 2019. ; https://doi.org/10.1101/634485doi: bioRxiv preprint

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Frequently Asked Questions (1)
Q1. What are the contributions mentioned in the paper "A cytochrome p450 from juvenile mustard leaf beetles hydroxylates geraniol, a key step in iridoid biosynthesis" ?

In the fat body, 73 individual cytochrome P450s were 43 certified by peer review ) is the author/funder.