Download CYP74C3 and CYP74A1, plant cytochrome P450 enzymes whose

8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology
CYP74C3 and CYP74A1, plant cytochrome P450
enzymes whose activity is regulated by detergent
micelle association, and proposed new rules for
the classification of CYP74 enzymes
R.K. Hughes1 , E.J. Belfield and R. Casey
John Innes Centre, Norwich Research Park, Norwich NR4 7UH, U.K.
Abstract
CYP74C3 (cytochrome P450 subfamily 74C3), an HPL (hydroperoxide lyase) from Medicago truncatula (barrel
medic), and CYP74A1, an AOS (allene oxide synthase) from Arabidopsis thaliana, are key membraneassociated P450 enzymes in plant oxylipin metabolism. Both recombinant detergent-free enzymes are
monomeric proteins with dual specificity and very low enzyme activity that can be massively activated
with detergent. This effect is a result of the formation of a complex between the protein monomer and a
single detergent micelle and, in the case of CYP74A1, has a major effect on the substrate specificity of the
enzyme. Association with a detergent micelle without an effect on protein oligomeric state represents a new
mechanism of activation for membrane-associated P450 enzymes. This may represent a second unifying
feature of CYP74 enzymes, in addition to their known differences in reaction mechanism, which separates
them functionally from more classical P450 enzymes. Highly concentrated and monodispersed samples of
detergent-free CYP74C3 and CYP74A1 proteins should be suitable for structural resolution. On the basis
of recent evidence for incorrect assignment of CYP74 function, using the current rules for CYP74 classification
based on sequence relatedness, we propose an alternative based on substrate and product specificity for
debate and discussion.
Introduction
Members of the CYP74 (cytochrome P450 subfamily 74) have
not been studied extensively. CYP74 enzymes are very different from classical P450 enzymes of microbial [1] or mammalian [2] origin, in that they have an atypical reaction mechanism that requires neither oxygen nor an NADPH-reductase
[3], and consequently have extremely high catalytic-centre
activities. In this sense, they have more in common with
non-classical mammalian P450 enzymes such as thromboxane synthase [4]. CYP74 enzymes all use fatty acid hydroperoxides as substrates and were originally classified on the
basis of their product specificity rather than sequence relatedness. Thus CYP74A refers to AOS (allene oxide synthase) and
CYP74B to HPL (hydroperoxide lyase) [5].
HPL cleaves hydroperoxides, formed from the oxygenation of PUFAs (polyunsaturated fatty acids) by the action of
LOX (lipoxygenase), into an array of volatile and non-volatile
products, some of which have antibacterial properties [6] and
so are important in plant defence. The volatile aldehydes are
Key words: allene oxide synthase, cytochrome P450 subfamily 74 (CYP74), detergent micelle,
haem, hydroperoxide lyase, oxylipin.
Abbreviations used: AOS, allene oxide synthase; CYP74, cytochrome P450 subfamily 74;
DES, divinyl ether synthase; Emulphogene, polyoxyethylene 10 tridecyl ether; HPL, hydroperoxide lyase; 9-HPODE, 9-S-hydroperoxyoctadeca-10E,12Z-dienoic acid; 13-HPODE, 13-Shydroperoxyoctadeca-9Z,11E-dienoic acid; 9-HPOTE, 9-S-hydroperoxyoctadeca-10E,12Z,15Ztrienoic acid; 13-HPOTE, 13-S-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid; LOX, lipoxygenase;
PUFA, polyunsaturated fatty acid; RZ, Reinheitszahl.
1
To whom correspondence should be addressed (email [email protected]).
of great value to the food industry [7] and are generated on
an industrial scale using vegetable oils rich in linoleic acid
and linolenic acid as a source of PUFA, defatted soya-bean
flour as a stable source of LOX and various sources of HPL
[8–10]. HPL is present at only very low levels in plants and
is relatively unstable [9], so the subsequent conversion of
the hydroperoxides into volatile aldehydes is usually carried
out by a recombinant HPL present in crude Escherichia coli
[8] or yeast [10] extracts; the latter would be more costly to
manufacture and the HPL activity in the crude E. coli extract
is stable only for 1 month at 4◦ C [8]. The use of a purified
HPL would seem to help matters and has been shown to
yield 20–85 times more aldehydes per gram of protein in a
shorter reaction time than the same protein in chloroplastenriched and crude plant extracts [9]. The stability of HPL
activity and protein from any source is evidently a current
problem in biocatalysis, but could be overcome to a large
extent by the development of a purified recombinant HPL
that remains stable and active in the longer term, both on the
shelf and during the reaction.
All HPLs are membrane-associated and require detergent
for extraction and solubilization. It has been difficult to
resolve the detergent and protein interactions, and consequently there is some disagreement about the oligomeric state
of HPL purified from a number of higher plants including,
for example, guava [11], bell pepper [9,12] and tomato [13].
The enzyme has been reported to be either trimeric or
C 2006
Biochemical Society
1223
1224
Biochemical Society Transactions (2006) Volume 34, part 6
tetrameric; the oligomeric state of various recombinant HPLs:
CYP74B1 [14], CYP74B2 [15], CYP74B3 [16], CYP74B4
[17], CYP74B5 [11], CYP74C1 [18] and CYP74C2 [19], and
the effects of detergent removal, are hardly ever reported. The
effects of detergent on increasing the activity of HPL are
well documented (see [20]) but the molecular mechanism
responsible for this activation were unknown. There is no
known mammalian equivalent of HPL.
HPL has the same substrate specificity as AOS. Unlike
HPL, which cleaves hydroperoxides, AOS transforms them
into unstable fatty acid epoxides which are then metabolized
further by enzymatic processes to jasmonates that are important in the signalling of plant defence responses; the mammalian equivalent of AOS is prostaglandin endoperoxide H
synthase [4]. With one exception, that of AOS purified from
flax seed [21], the oligomeric state of AOS purified from a
number of higher plants, including guayule [4,22] and corn
[23], and of recombinant AOSs from barley [24], tomato
[5,16] and Arabidopsis [25] has not been reported because,
like HPL, it has proved difficult to resolve the detergent and
protein interactions. Gel filtration analysis of AOS purified
from flax seed in the presence of detergent confirmed that the
protein remained as a monomer of molecular mass 55 kDa
[21], which suggested that there was no association with
detergent micelles and that the protein was entirely watersoluble. In the same work, however, it was reported that
the specific activity of the enzyme was enhanced 2–3-fold
by detergent, but the molecular mechanism responsible for
this activation is unknown. The molecular mechanisms and
primary determinants of the differences in CYP74 product
specificities are also unknown, primarily because to date
there is no published structure of any plant cytochrome P450
enzyme.
Detergent micelles have been shown to modify the activities of classical P450 enzymes through an effect on oligomeric
state. Thus, in the presence of 10 mM n-octyl glucoside, a
complete loss of catalytic activity of CYP2B4 with reductase
was observed due to disaggregation of the active pentamer
or hexamer into inactive monomers [26]. The effects of the
detergent Emulphogene (polyoxyethylene 10 tridecyl ether)
on CYP2B4 (and CYP1A2) activity are, however, contradictory (see [27]) with reports of both loss and gain of catalytic
activity due to the formation of monomers or dimers respectively.
We have expressed in milligram quantities and fully characterized two plant cytochrome P450 enzymes: CYP74C3
(an HPL from Medicago truncatula) [28] and CYP74A1 (an
AOS from Arabidopsis thaliana) [29] in the presence and
absence of detergent. We first present new and unexpected
information on their substrate and product specificities and
oligomeric states. Secondly, we describe the ability of both
proteins to exhibit activation kinetics through association
with a micelle of Emulphogene without an apparent change in
oligomeric state. Thirdly, we discuss the production of highly
concentrated and monodispersed samples of detergent-free
CYP74C3 and CYP74A1 proteins which may be well suited
for the purposes of crystallization and structural resolution.
C 2006
Biochemical Society
Finally, reasons are outlined for the introduction of a new
classification system for CYP74 enzymes based on function
rather than sequence relatedness.
CYP74C3 and CYP74A1 are monomeric
proteins with dual specificity
Analysis of the oligomeric state of CYP74A1 (Figure 1a)
and CYP74C3 (Figure 1b) in the absence of detergent
has demonstrated for the first time that both CYP74
enzymes are highly water-soluble and monomeric proteins of
molecular mass approx. 55 kDa. The enzymes were purified
to homogeneity [RZ > 1.3; CYP74C3 [28]; and RZ > 1.1;
CYP74A1 [29]; where RZ is Reinheitszahl (or purity index)]
with a full complement of haem iron. The catalytic efficiency
(kcat /K m ) of the detergent-free proteins (Table 1) compared
with the same proteins isolated without detergent removal
[28,29] was, however, very low and suggested that the presence of detergent micelles was essential to maintain the most
active conformation of both proteins.
Phylogenetic analysis of CYP74C3 suggested that it
had dual specificity [6] and this was confirmed by GC–
MS analysis of the products in reactions of the purified
recombinant protein with 9-HPODE (9-S-hydroperoxyoctadeca-10E,12Z-dienoic acid), 9-HPOTE (9-S-hydroperoxyoctadeca-10E,12Z,15Z-trienoic acid), 13-HPODE
(13-S-hydroperoxyoctadeca-9Z,11E-dienoic acid) and 13HPOTE (13-S-hydroperoxyoctadeca-9Z,11E,15Z-trienoic
acid) [28]. Phylogenetic analysis of CYP74A1 indicated that
it was a monospecific 13-AOS [5], and this was apparently
confirmed in a previous work using recombinant enzyme
[25], although no comments on activities with 9-HPODE or
9-HPOTE were reported in this study. Recent work in our
laboratory has revealed, however, that CYP74A1 has major
activity with 9-HPODE and 9-HPOTE and exhibits dual
specificity [29].
The biological activity of CYP74C3 and
CYP74A1 is regulated by monomer–micelle
association
Analysis by gel filtration in the presence of detergent micelles
of the oligomeric states of CYP74A1 and CYP74C3 indicated
that CYP74A1 formed almost entirely a complex of approx.
110 kDa (Figure 1c); CYP74C3 formed a number of higher
oligomers, but the most active species was a complex of
similar size to that formed by CYP74A1 (Figure 1d). Both
these complexes were identified as a protein monomer
complexed with a single detergent micelle of molecular mass
approx. 62 kDa [28,29]. Micelle binding increased the kcat /K m
of CYP74C3 and CYP74A1 with the preferred substrate 13HPOTE by approx. 50-fold (Table 1). The increase in kcat /K m
of up to 1.6 × 108 and 5.9 × 107 M−1 · s−1 for CYP74C3 and
CYP74A1 respectively is especially remarkable for CYP74C3
whose reaction mechanism involves the scission of a C–C
bond. Micelle-induced changes in the catalytic efficiencies of
both proteins were accompanied by a shift in equilibrium
8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology
Figure 1 CYP74C3 and CYP74A1 are monomeric proteins that bind a single detergent micelle
Purified CYP74A1 or CYP74C3 was loaded on to calibrated Superdex 200 16/60 or Superdex 75 26/60 columns respectively
in the absence (a, b) or presence (c, d) of Emulphogene. Both detergent-free proteins eluted almost exclusively at sizes
corresponding to the protein monomers (∼55 kDa). In the presence of detergent, the peak position of CYP74A1 protein
shifted to that corresponding to a complex of molecular mass approx. 110 kDa; in addition, the peak position of CYP74C3
protein shifted to a number of species of higher mass. The peak positions of CYP74A1 and CYP74C3 activities corresponded
to the size of a monomer–micelle complex.
Table 1 Activation of CYP74C3 and CYP74A1
CYP74C3
Kinetic parameter
k cat /K m (µM−1 · s−1 )†
13-HPOTE
13-HPODE
9-HPOTE
9-HPODE
Substrate specificity (%)
13-HPOTE
13-HPODE
9-HPOTE
Detergent-free
CYP74A1
Activated*
Detergent-free
Activated*
3.2
0.6
149.3
33.5
1.2
0.9
58.7
8.1
0.1
0.5
0.6
7.2
0.3
0.8
0.2
0.2
100
19
3
100
22
0.4
100
75
100
14
25
0.3
*With 50 µM Emulphogene micelle.
†See [28,29] for individual K m and k cat values. Assumes one active site per monomer of molecular mass 56.8 or 55.3 kDa for CYP74C3 and CYP74A1
respectively.
towards low-spin haem iron, which was confirmed by UV–
visible and EPR spectroscopy [28,29]. An analysis of micelle
binding to both detergent-free proteins indicated that the
detergent micelle bound very tightly to both protein monomers with K d values of 6.9 ± 1.1 and 10.7 ± 1.7 µM for
CYP74C3 and CYP74A1 respectively, which are similar or
tighter than the preferred substrate 13-HPOTE [28,29]. The
substrate specificity of CYP74A1 was greatly modified upon
micelle binding to one that had considerably more 13-AOS
activity, but the level of 9-AOS activity was not insignificant
C 2006
Biochemical Society
1225
1226
Biochemical Society Transactions (2006) Volume 34, part 6
and CYP74A1 would still correctly be described as exhibiting
dual specificity even in the micelle-bound form.
Production of CYP74C3 and CYP74A1 for
structural resolution
To date, no structure has been published for any plant cytochrome P450 or CYP74 enzyme. The structure of the AOS
domain of an AOS–LOX chimaeric protein from coral has
been solved and shown to be very similar to a catalase [30],
but this is highly dissimilar to HPLs, which are neither watersoluble in their most active state nor predicted to have a
catalase-fold. CYP74C3 and CYP74A1 have been purified
from E. coli to produce detergent-free proteins that are
almost entirely monomeric, monodisperse and water-soluble
to 50 mg/ml protein, based on haem concentration. Crystallization trials using both oil immersion and hanging drop techniques have been conducted in three different laboratories
with relatively little success. No crystals of CYP74C3
have been obtained and the fragile, needle-like crystals of
CYP74A1 that were obtained were unsuitable for X-ray
diffraction. We have now carried out modelling and mutagenesis experiments to successfully identify mutants of
CYP74C3 that are modified in both substrate and micelle
binding, the first to be identified for a CYP74 enzyme and
which may prove to be more amenable to crystallization.
Proposed new rules for the classification
of CYP74 enzymes
We suggest a new scheme for CYP74 classification. Since
the discovery of the first CYP74 enzyme, an AOS purified
from flax seed [3], the classification of CYP74 enzymes
has been relatively straightforward and based on sequence
relatedness [31]. Fortunately, this also separated them on the
basis of function. Thus CYP74A and CYP74B corresponded
to AOS and HPL respectively. The substrate specificity of
CYP74A and CYP74B enzymes was previously shown to be
monospecific and restricted to either 9- or 13-hydroperoxide
substrates. The discovery of new HPL [19] and AOS [5]
enzymes with dual specificity, turning over both 9- and
13-hydroperoxides, has since led to the emergence of a
third group, CYP74C, which contains any CYP74 with
dual specificity and is unrelated to product specificity. The
recent revelation that recombinant AOS from Arabidopsis
(CYP74A1) has dual specificity [29], despite its identification
as a 13-AOS based on sequence relatedness, would suggest
that CYP74 assignation should be based on evidence of
both substrate and product specificity. Moreover, CYP74
designation should only be applied to those sequences
that are proven to encode active recombinant proteins and
whose substrate and product specificities have been fully
determined using purified protein in vitro. The current use
of CYP74 classification for unpublished sequences, or where
the true function has not been verified, is both misleading
and premature. Under the proposed new scheme, CYP74C
would include only HPLs with dual specificity: CYP74C1
(cucumber HPL [18]), CYP74C2 (melon HPL [19]) and
C 2006
Biochemical Society
CYP74C3 (barrel medic HPL [28]). CYP74D would be
assigned to AOS with dual specificity. For the purposes of
this discussion, AOS from Arabidopsis remains classified as
CYP74A1, but would be reclassified as CYP74D4 in the new
scheme: CYP74D1 (tomato AOS [5], previously classified
as CYP74C3) and CYP74D2 (barley AOS [24]). A new
CYP74C with dual specificity has very recently been described in potato [32] and would be reclassified as CYP74D3
in the new scheme. CYP74E would be assigned to DES
(divinyl ether synthase): CYP74E1 (tomato DES [33]). New
sequences from rice in the P450 database that are currently
classified as CYP74E and CYP74F (http://drnelson.utmem.
edu/CytochromeP450.html) are unpublished so would be
excluded from this classification scheme. Reclassification
based on function is a necessary pre-requisite for the interpretation of the roles of CYP74 enzymes in plant oxylipin
metabolism. We respectfully offer our new proposal to
the nomenclature committee on P450 classification and the
CYP74 scientific community as a basis for debate and
discussion.
We thank Professor Mats Hamberg (Karolinska Institute, Stockholm,
Sweden) for providing CYP74 substrates. This work was funded by a
European Union-funded project ‘NODO (Natural Oxylipins for Defence
of Ornamentals)’, project number QLK5-CT-2001-02445, and by the
Biotechnology and Biological Sciences Research Council.
References
1 Hasemann, C.A., Kurumbail, R.G., Boddupalli, S.S., Peterson, J.A. and
Desienhofer, J. (1995) Structure 2, 41–62
2 Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F. and McRee, D.E. (2000)
Mol. Cell 5, 121–131
3 Song, W.-C., Funk, C.D. and Brash, A.R. (1993) Proc. Natl. Acad. Sci. U.S.A.
90, 8519–8523
4 Pan, Z., Camara, B., Gardner, H.W. and Backhaus, R.A. (1998)
J. Biol. Chem. 273, 18139–18145
5 Itoh, A., Schilmiller, A.L., McCaig, B.C. and Howe, G.A. (2002)
J. Biol. Chem. 277, 46051–46058
6 Feussner, I. and Wasternack, C. (2002) Annu. Rev. Plant Physiol.
Plant Mol. Biol. 53, 275–297
7 Casey, R. and Hughes, R.K. (2004) Food Biotechnol. 18, 135–170
8 Noordermeer, M.A., van der Goot, W., van Kooij, A.J., Veldsink, J.W.,
Veldink, G.A. and Vliegenthart, J.F.G. (2002) J. Agric. Food Chem. 50,
4270–4274
9 Husson, F. and Belin, J.M. (2002) J. Agric. Food Chem. 50, 1991–1995
10 Häusler, A., Lerch, K., Muheim, A. and Silke, N. (2001) U.S. Patent
6238898
11 Tijet, N., Waspi, U., Gaskin, D.J., Hunziker, P., Muller, B.L., Vulfson, E.N.,
Slusarenko, A., Brash, A.R. and Whitehead, I.M. (2000) Lipids 35,
709–720
12 Shibata, Y., Matsui, K., Kajiwara, T. and Hatanaka, A. (1995)
Plant Cell Physiol. 36, 147–156
13 Fauconnier, M.-L., Perez, A.G., Sanz, C. and Marlier, M. (1997)
J. Agric. Food Chem. 45, 4232–4236
14 Psylinakis, E., Davoras, E.M., Ioannidis, N., Trikeriotis, M., Petrouleas, V.
and Ghanotakis, D.F. (2001) Biochim. Biophys. Acta 1533, 119–127
15 Kandzia, R., Stumpe, M., Berndt, E., Szalata, M., Matsui, K. and
Feussner, I. (2003) J. Plant Physiol. 160, 803–809
16 Howe, G.A., Lee, G.I., Itoh, A., Li, L. and DeRocher, A.E. (2000)
Plant Physiol. 123, 711–724
17 Noordermeer, M.A., van Dijken, A.J.H., Smeekens, S.C.M., Veldink, G.A.
and Vliegenthart, J.F.G. (2000) Eur. J. Biochem. 267, 2473–2482
8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology
18 Matsui, K., Ujita, C., Fujimoto, S.-H., Wilkinson, J., Hiatt, B., Knauf, V.,
Kajiwara, T. and Feussner, I. (2000) FEBS Lett. 481, 183–188
19 Tijet, N., Schneider, C., Muller, B.L. and Brash, A.R. (2001)
Arch. Biochem. Biophys. 386, 281–289
20 Koeduka, T., Stumpe, M., Matsui, K., Kajiwara, T. and Feussner, I. (2003)
Lipids 38, 1167–1172
21 Song, W.-C. and Brash, A.R. (1991) Science 253, 781–784
22 Pan, Z., Durst, F., Werck-Reichhart, D., Gardner, H.W., Camara, B.,
Cornish, K. and Backhaus, R.A. (1995) J. Biol. Chem. 270, 8487–8494
23 Utsunomiya, Y., Nakayama, T., Oohira, H., Hirota, R., Mori, T., Kawai, F.
and Ueda, T. (2000) Phytochemistry 53, 319–323
24 Maucher, H., Hause, B., Feussner, I., Ziegler, J. and Wasternack, C. (2000)
Plant J. 21, 199–213
25 Laudert, D.L., Pfannschmidt, U., Lottspeich, F., Hollander-Czytko, H. and
Weiler, E.W. (1996) Plant Mol. Biol. 31, 323–335
26 Dean, W.L. and Gray, R.D. (1982) J. Biol. Chem. 257, 14679–14685
27 Viner, R.I., Novikov, K.N., Ritov, V.B., Kagan, V.E. and Alterman, M.A.
(1995) Biochem. Biophys. Res. Commun. 217, 886–891
28 Hughes, R.K., Belfield, E.J., Muthusamay, M., Khan, A., Rowe, A.,
Harding, S.E., Fairhurst, S.A., Bornemann, S., Ashton, R., Thorneley, R.N.F.
and Casey, R. (2006) Biochem. J. 395, 641–652
29 Hughes, R.K., Belfield, E.J., Ashton, R., Fairhurst, S.A., Göbel, C.,
Feussner, I. and Casey, R. (2006) FEBS Lett. 580, 4189–4195
30 Oldham, M.L., Brash, A.R. and Newcomer, M.E. (2005) Proc. Natl.
Acad. Sci. U.S.A. 102, 297–302
31 Nelson, D.R. (2002) Methods Enzymol. 357, 13–15
32 Stumpe, M., Göbel, C., Demchenko, K., Hoffmann, M., Klösgen, R.B.,
Pawlowski, K. and Feussner, I. (2006) Plant J. 47, 883–896
33 Itoh, A. and Howe, G.A. (2001) J. Biol. Chem. 276, 3620–3627
Received 26 June 2006
C 2006
Biochemical Society
1227