1Department of Cell and Structural Biology and 2School of Chemical Sciences, University of Illinois, Urbana, IL 61801, USA
3 To whom correspondence should be addressed. e-mail: maryschu{at}uiuc.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: homology modeling/P450/P450 monooxygenases/phenylpropanoid pathway/substrate docking
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Since the cloning of the first plant P450 in 1990 (Bozak et al., 1990), substantial work has been done on the cloning and characterization of a large array of P450s involved in essential and non-essential plant biochemistries (Schuler, 1996
; Chapple, 1998
; Werck-Reichhart et al., 2002
; Schuler and Werck-Reichhart, 2003
). P450s are found in all major plant biosynthetic pathways, including those for flavonoids, anthocyanins, phenylpropanoids, terpenoids, alkaloids, cyanogenic glycosides, fatty acids, hormones and signaling molecules (Werck-Reichhart, 1995
). Analysis at the primary sequence level of these many P450s with perspectives on evolution (Kahn and Durst, 2000
) has suggested that divergence of these enzymes allows for the acquisition of new biochemical reactivities and/or sets of reactivities that have potential for evolving new biochemical pathways. Among closely related P450s, one clear example of this is the set of four maize and four wheat CYP71C subfamily proteins that mediate consecutive steps in the synthesis of DIMBOA, a defense agent against fungal and insect pests (Frey et al., 1997
; Nomura et al., 2002
). Another example is the set of Arabidopsis CYP90 family proteins that mediate consecutive steps in brassinolide synthesis (Szekeres et al., 1996
; Choe et al., 1998
).
Even so, there are perhaps more examples of biochemical pathways containing P450s categorized in very different families that are capable of handling similar substrates. Examples of these are the Arabidopsis CYP701A3 and redundant CYP88A3/CYP88A4 proteins involved in gibberellin synthesis (Helliwell et al., 1998, 1999, 2001). Others are the Arabidopsis CYP86A and Vicia CYP94A subfamily proteins that mediate
-hydroxylations on C12 fatty acids (Benveniste et al., 1998
; Tijet et al., 1998
; Le Bouquin et al., 1999
; Wellesen et al., 2001
). Within Arabidopsis, additional examples exist in the CYP73A5, CYP75B1, CYP84A1 and CYP98A3 proteins that mediate various steps in phenylpropanoid biosynthesis (Figure 1), including one in the core pathway, two in lignin synthesis and one in flavonoid/anthocyanin synthesis (Mizutani et al., 1997
; Urban et al., 1997
; Humphreys et al., 1999
; Schoenbohm et al., 2000
; Schoch et al., 2001
; Franke et al., 2002
). In this last example, CYP73A5 (t-cinnamic acid hydroxylase, t-CAH) catalyzes the 4-hydroxylation of t-cinnamate to p-coumarate (Figure 1). CYP84A1 catalyzes the 5-hydroxylation of the similarly sized coniferaldehyde, coniferyl alcohol and ferulic acid, CYP75B1 catalyzes the 3-hydroxylation of the larger dihydrokaempferol structure and CYP98A3, which represents the newest member of this collection of phenylpropanoid enzymes, catalyzes the 3-hydroxylation of the larger p-coumaroylshikimic and quinic acids. Given their high degree of sequence divergence, it has been unclear whether the catalytic sites of these divergent proteins maintain side chain conservations important for interactions with conserved features of their phenylpropanoid substrates.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the initial modeling of each P450 target sequence, 10 models were generated for each P450 with the explicit inclusion of heme coordinates in all steps of homology model generation. These models were subjected to coarse energy minimization procedures in order to remove potentially bad van der Waals contacts between atoms. In the subsequent modeling, the best model ranked by MOEs residue packing quality function was selected and the heme coordinates were copied from the CYP102 crystal structure with a covalent bond created between the hemes iron atom and the sulfur of the conserved cysteine axial ligand. Further energy minimizations for each protein were performed using the CHARMm22 force field (MacKerell et al., 1998) within the MOE distribution until the final energy gradient was <0.01 kcal/mol.Å. A distance-dependent dielectric constant was used in the calculations with a cutoff between 6.5 and 7 Å.
Known substrates for each of the Arabidopsis P450s were docked within the catalytic site of the energy-minimized model using the Monte Carlo docking procedure of MOE after attaching a single oxygen to the heme plane (representing the ironoxo intermediate). Parameters for the FO bond and NFeO angle were obtained using MOEs parameter assignment facility and are listed in Table I in CHARMm notation. In this docking, each substrate was initially placed above the heme plane and allowed to vary through Monte Carlo simulations removing any bias due to manual placement. Twenty-five possible conformations were generated for each substrate while maintaining rigid side chains and these were ranked according to the sum of the ligands internal energy and the van der Waals and electrostatic energy terms of the potential energy function. The binding conformation with the lowest energy and appropriate hydroxylation site closest to the heme was selected as the optimal conformation and subjected to energy minimization using the MMFF94 force field (Halgren, 1996) in MOE while allowing full side chain relaxation. In these protein/ligand minimizations, the heme coordinates were fixed to prevent distortion of the heme plane originating from bonded parameters in the MOEs implementation of the MMFF94 force field.
|
The final Arabidopsis P450 models were subjected to several tests to assess their reliability. The first test was to examine the distribution of and
angles using Ramachandran plots generated within the MOE program. The second test was to apply energy criteria using Prosa II (version 3.02) analysis (Center for Applied Molecular Engineering, University of Salzburg, Austria). In this program, the Prosa Z-scores and energy profiles were calculated for each model in order to assess their reliability. The third test was to calculate residue compatibility profiles (3D1D score) and the sum of residue compatibility scores for each model using Profiles 3D analysis (InsightII Homology module, MSI, San Diego, CA). The fourth test was to determine the thermodynamic and structural stability of the models during 200 ps unconstrained molecular dynamics (MD) simulations of the unbound enzyme using CHARMm22 force fields (MacKerell et al., 1998
) within the MOE program. MD simulations were run in the absence of explicit water molecules but with a distance-dependent dielectric constant. The canonical ensemble (NVT) was used with a target temperature of 300 K. An integration time step of 1 fs was used and structures were saved to disk every 100 steps.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of comparisons of the crystal structures of seven bacterial P450s and a mammalian P450 have now indicated that, despite a relatively low degree of sequence identity (only 13%) and significant size heterogeneity (4562 kDa), their protein architectures are remarkably conserved (Hasemann et al. 1995; Peterson and Graham-Lorence, 1995
; Williams et al., 2000
). The conserved architecture of the P450 crystal structures consists of four to five ß-pleated sheets (designated 15) and 15
-helixes (designated AL). Overall, the structure is classified into an
-domain that contains most of the
-helixes with three small ß-pleated sheets and a ß-domain that contains the larger ß1- and ß2-sheets with three
-helixes. Structural overlays of the C
backbones of four bacterial P450 crystal structures [P450cam (CYP101) (Poulos et al., 1987
), P450BM3 (CYP102) (Ravichandran et al., 1993
; Li and Poulos, 1994
, 1997
), P450eryF (CYP107A1) (Cupp-Vickery and Poulos, 1995
), P450terp (CYP108) (Hasemann et al., 1994
)] and one mammalian P450 (CYP2C5) (Williams et al., 2000
) have indicated, as shown in Figure 2, that >50% of each proteinss backbone occurs within an r.m.s.d. of 2 Å and that at least 90% of the backbone occurs within an r.m.s.d. of 5 Å. The most conserved structural elements with respect to their length and relative C
positions are ß-pleated sheets 1, 4 and 5 and
-helixes D, E, I, K and L. In addition, the C
backbones of the loop between helix K and strand 4 of ß-pleated sheet 1 (ß-sheet 14), the heme binding loop and the loop between helices L and K' (designated as the meander region) are highly conserved. Conserved residues shared by all of these P450s are those in the P450 signature motif (FGRCG) that contains the heme binding cysteine and the (E/D)T pair in helix I that mediates dioxygen activation through a distal charge relay system (Atkins and Sligar, 1988
; Aikens and Sligar, 1994
).
|
As detailed in Materials and methods, the four phenylpropanoid pathway P450s from Arabidopsis thaliana were modeled using the CYP102 structure as the template. The four Arabidopsis P450s share 15.817.5% sequence identity with this bacterial P450 compared with 22.524.3% sequence identity with the eukaryotic CYP2C5 protein. The CYP102 structure was chosen as template because evaluation of the crystal structures with Prosa II indicated some high-energy regions in CYP2C5 structure. Profiles 3D, 3D1D self-compatibility scores (Table IV) suggest that some residues in the structure are in incompatible environments, including residues 209236 and 342346 (corresponding to residues 179206 and 312316 in Figure 4). CYP2C5s Prosa II normalized Z-score (Table III) is the lowest among all crystal structures investigated here, as has also been reported recently (Kirton et al., 2002).
Using the CYP102 structural coordinates, 10 models were generated for each P450 protein with the explicit inclusion of the heme coordinates in all steps of homology model generation. After subjecting all of these models to coarse energy minimizations that eliminate bad van der Waals contacts between atoms, the best-ranked model was further minimized using the CHARMm22 force field within the MOE program (version 2002) until the final energy gradient was <0.01 kcal/mol.Å.
All four of the final models display considerable similarity in the -domain with the P450 core structure shown in Figure 2. Specifically, all contain a well-defined A-helix with 13 turns, a B-helix with 12 turns, a B'-helix with 13 turns, an E-helix with 45 turns, a G-helix with 58 turns, a J-helix with 45 turns and a K-helix with 34 turns. Some variability occurs between the models in the lengths of their D-helix (25 turns), F-helix (34 turns) and L-helix (25 turns). Additionally, the CYP73A5 and CYP75B1 models predict a kink in the middle of the I-helix at amino acids Ala306 and Gly303, respectively. At this level of comparison, all four P450 models contain a structurally conserved five-stranded ß1-sheet and more variability in the remaining three ß-pleated sheets of their ß-domains. Specifically, the CYP75B1 and CYP98A3 models contain an intact two-stranded ß2-sheet that is not present in the CYP73A5 and CYP84A1 models. The CYP73A5 model contains an intact three-stranded ß3-sheet, the CYP84A1 model contains the second and third strands of a ß3-sheet and neither the CYP75B1 nor the CYP98A3 model contains a ß3-sheet. The CYP75B1 and CYP98A3 models contain a two-stranded ß4-sheet, the CYP73A5 model contains a shortened ß4-sheet and the CYP84A1 model lacks all strands of this ß-sheet.
Quality of the models
To investigate the quality of these substrate-free Arabidopsis P450 models, a variety of tests described in Materials and methods were performed. The first of these tests used Ramachandran plots to analyze the and
angle distributions for each model (Table II). This analysis demonstrates that all the models display more than 97% of their residues (excluding Gly and Pro residues) in allowed areas of the Ramachandran map and indicates that all four of these models are of a quality similar to those published for other bacterial P450 models (Chang and Loew, 1996
, 2000; Chang et al., 1997
).
|
|
|
|
|
|
Substrates (Figure 1) were docked within the active site of each P450 using the Monte Carlo docking procedures within the MOE program and repeated cycles of protein and substrate minimization as described in Materials and methods. Of the 25 different conformations obtained in this substrate docking procedure, the lowest energy conformation having the substrate hydroxylation site positioned in closest proximity to the heme-bound oxygen was selected as the most probable binding mode. This docking mode was subjected to energy minimization using the MMFF94 force field in MOE, allowing full side chain relaxation while keeping the heme coordinates fixed to prevent distortion of the heme plane originating from bonded parameters in the MOEs implementation of the MMFF94 force field.
Examination of the selected binding conformations of all four models indicates definite similarities in the orientations (Figure 6). In all four models, the aromatic ring of each ligand is positioned above the protoporphyrin IX heme at an angle ranging from 53 to 83° relative to the heme, two of these models have their ligands axis (defined by the non-reacting region) positioned above the heme rings III and I and two (CYP73A5 and CYP98A3) have the ligand positioned above heme rings III and II. Additional similarities between the binding modes are apparent in the positioning of the substrates relative to the C backbone of helix I, which traverses the catalytic site above heme rings I and IV (Figure 6). In all of these predicted binding modes, the aromatic ring region is oriented toward the C-terminus of helix I and the variable length tail is oriented toward its N-terminus. In this orientation, the distance between the ironoxo intermediate and the conserved Thr in helix I presumed, by analogy to other P450s (Atkins and Sligar, 1988
; Aikens and Sligar, 1994
), to be involved in oxygen activation is 4.4 Å for Thr310 in CYP73A5, 4.3 Å for Thr323 in CYP84A1, 4.6 Å for Thr303 in CYP98A3 and 5.1 Å for Thr306 in CYP75B1. As shown in Figure 7, which depicts only those amino acids lying within 4 Å of each substrate, these orientations place the aromatic rings of all substrates in proximity to amino acids in SRS4, SRS5 and SRS6 and the variable tails in proximity to amino acids in SRS1 and SRS2 (Figure 7).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the numerous methods used to analyze the quality of the predicted structures, all indicate that, overall, the models are well folded and thermodynamically stable. Some test results, such as those obtained for and
angle distributions in Ramachandran plots, indicate a small number of residues existing outside allowed angles. Examination of individual models indicates that these residues typically exist outside predicted SRS regions defining catalytic site specificities in various P450s. Other test results, such as the normalized Z-scores, which fall slightly below the recommended score of 0.7 generated in the Prosa II analysis or the S-scores generated in the Profiles 3D analysis, suggest the possibility of short regions of high energy or residues being in incompatible environments but, again, all of these residues are located outside the predicted SRS regions. More telling MD simulations indicate that these substrate-free P450 models are thermodynamically stable with r.m.s.d. and radius of gyration reaching equilibrium within 5060 ps of the start of the simulation and the temperature of the system remaining constant throughout each simulation.
Substrate docking experiments indicate that, although these four P450s share only 13% amino acid identity, similar mechanisms for substrate binding are predicted for all of these proteins. Specifically, all of these P450s display optimal binding modes that position the aromatic rings of their substrates at a 5383° angle to the heme plane with the hydroxylation site positioned at a distance of 2.43.6 Å to the oxygen of the ironoxo intermediate. In all four of the lowest energy binding conformations, the axis of the substrates aligns parallel to helix I with the aromatic ring oriented towards its C-terminus and the remainder of the molecule oriented towards the N-terminal end.
In all four models, all of the residues having at least one atom within 4 Å of the reactive ring are located in the C-terminal end of the SRS4 region or in the SRS5 and SRS6 regions (Figure 7). Conversely, all of the residues having at least one atom within 4 Å from the aliphatic end of each substrate are located in the SRS1 and SRS2 regions and the N-terminal end of the SRS4 region. These results suggest that the SRS5 and SRS6 regions and the C-terminal end of the SRS4 region are important in contacting the aromatic rings of these substrates and that the SRS1 and SRS2 regions and the N-terminal end of the SRS4 region are important in contacting the aliphatic regions of these substrates. It is worth noting that on the larger substrates, including dihydrokaempferol and p-coumaroylshikimic acid, contacts with SRS4 are not more extended than on the smaller substrates, all SRS4 interactions terminate at approximately the position of the carboxylic acid group on t-cinnamate or the aldehyde group on coniferaldehyde.
Comparison with the available substrate-bound CYP102 crystal structure (Li and Poulos, 1997) indicates that the Arabidopsis P450 substrate binding modes are different from the CYP102 substrate binding mode in which palmitoleic acid is positioned nearly perpendicular to helix I and over heme rings II and III (Figure 8). Comparisons of substrate positionings with other available substrate-bound P450 crystal structures are difficult because many of these substrates bear little resemblance to the linear cinnamate derivatives examined here. Notably, our predicted substrate binding mode for t-cinnamate in the Arabidopsis CYP73A5 catalytic site is consistent with NMR data showing that t-cinnamate binds parallel to helix I in the Helianthus tuberosus CYP73A1 protein (D.Werck-Reichhart, personal communication).
|
|
By comparison, other SRS regions of these P450s display significantly fewer absolute sequence identities (Figure 9). The SRS4 region contains a wide variety of residues that are not conserved in simple primary sequence alignments of these four divergent P450s, in contrast to comparisons of P450s done with the same subfamily. Despite these variations, there are clear conservations in the length and geometry of helix I in all four proteins and in their pattern of hydrophobic, charged and specific residues within the conserved hhchhAGhaThA sequence (where lower case c is a charged residue and lower case a is an acidic residue). Within this sequence, several closely aligned residues are predicted to contact the substrate in all four proteins. These include the conserved (Asp/Glu)Thr pair of residues that align with Asp251 and Thr252 in bacterial P450cam that mediate dioxygen activation (Atkins and Sligar, 1988; Aikens and Sligar, 1994
), the charged Asp/Asn residue near the N-terminus of helix I and the (Ala/Gly)(Ala/Gly) pair in the center of helix I.
The SRS6 region contains an MahGLh sequence that is conserved in the CYP84A1, CYP75B1 and CYP98A3 proteins and divergent in the CYP73A5 protein. Another residue N-terminal to the conserved Leu is also predicted to contact the substrate in three models except in CYP98A3 where a residue C-terminal to the Leu is predicted to make contacts. Among these, the hydrophobic Leu aligns with Phe494 in Vicia CYP94A2 that is critical for -fatty acid hydroxylation (Kahn et al., 2001
).
Relative to these other SRS regions, the SRS2 region lacks sequence conservations and the SRS1 region has relatively few. This is understandable in the light of their predicted roles in contacting the diversified tails on these substrates that vary significantly in their size. Even the CYP73A5 and CYP84A1 proteins that have similarly sized groups contacting this region exhibit little conservation in the identity of their contact residues apart from the aromatic residue in SRS2 that contacts both substrates.
In conclusion, these results indicate a common substrate-recognition mechanism among these four P450 proteins that involves similarities in (i) the orientation of the substrate relative to the C backbone and heme group of the models, (ii) the localization of residues in SRS regions contacting the substrate, and (iii) the sequence similarities of the predicted SRS regions. Future site-directed mutagenesis on these proteins will clarify the roles of individual amino acids in this substrate recognition process and the flexibility of each catalytic site in accepting alternative substrates.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Atkins,W.M. and Sligar,S.G. (1988) J. Biol. Chem., 263, 1884218849.
Benveniste,I., Tijet,N., Adas,F., Philipps,G., Salaün,J.-P. and Durst,F. (1998) Biochem. Biophys. Res. Commun., 243, 688693.[CrossRef][ISI][Medline]
Berman,H.M., Westbrook,J., Feng,Z., Gilliland,G., Bhat,T.N., Weissig,H., Shindyalov,I.N. and Bourne,P.E. (2000) Nucleic Acids Res., 28, 235242.
Bozak,K.R., Yu,H., Sirevag,R. and Christoffersen,R.E. (1990) Proc. Natl Acad. Sci. USA, 87, 39043908.[Abstract]
Chang,Y.T. and Loew,G.H. (1996) Protein Eng., 9, 755766.[Abstract]
Chang,Y.T. and Loew,G.H. (2000) Biochemistry, 39, 24842498.[CrossRef][ISI][Medline]
Chang,Y.T., Stiffelman,O.B., Vakser,I.A., Loew,G.H., Bridges,A. and Waskell,L. (1997) Protein Eng., 10, 119129.[Abstract]
Chapple,C. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol., 49, 311343.[CrossRef][ISI]
Choe,S., Dilkes,B.P., Fujioka,S., Takatsuto,S., Sakurai,A. and Feldmann,K.A. (1998) Plant Cell, 10, 231243.
Cupp-Vickery,J.R. and Poulos,T.L. (1995) Nat. Struct. Biol., 2, 144153.[ISI][Medline]
Franke,R., Humphreys,J.M., Hemm,M.R., Denault,J.W., Ruegger,M.O., Cusumano,J.C. and Chapple,C. (2002) Plant J., 30, 3345.[CrossRef][ISI][Medline]
Frey,M. et al. (1997) Science, 277, 696699.
Gotoh,O. (1992) J. Biol. Chem., 267, 8390.
Halgren,T.A. (1996) J. Comput. Chem., 17, 490.[CrossRef][ISI]
Hasemann,C.A., Kurmbail,R.G., Peterson,J.A. and Deisenhofer,J. (1994) J. Mol. Biol., 236, 11691185.[ISI][Medline]
Hasemann,C.A., Kurmbail,R.G., Boddupalli,S.S., Peterson,J.A. and Deisenhofer,J. (1995) Structure, 3, 4162.[ISI][Medline]
Helliwell,C.A., Sheldon,C.C., Olive,M.R., Walker,A.R., Zeevaart,J.A., Peacock,W.J. and Dennis,E.S. (1998) Proc. Natl Acad. Sci. USA, 95, 90199024.
Helliwell,C.A., Poole,A., Peacock,W.A. and Dennis,E.S. (1999) Plant Physiol., 119, 507510.
Helliwell,C.A., Chandler,P.M., Poole,A., Dennis,E.S. and Peacock,W.J. (2001) Proc. Natl Acad. Sci. USA, 98, 20652070.
Humphreys,J.M., Hemm,M.R. and Chapple,C. (1999) Proc. Natl Acad. Sci. USA, 96, 1004510050.
Kahn,R.A. and Durst,F. (2000) Recent Adv. Phytochem., 34, 151189.
Kahn,R.A., Le Bouquin,R., Pinot,F., Benveniste,I. and Durst,F. (2001) Arch. Biochem. Biophys., 391, 180187.[CrossRef][ISI][Medline]
Karplus,P.A. (1997) Protein Sci., 6, 13021307.
Kirton,S.B., Kemp,C.A., Tomkinson,N.P., St-Gallay,S. and Sutcliffe,M.J. (2002). Proteins: Struct. Funct. Genet., 59, 216231.[CrossRef]
Le Bouquin,R., Pinot,F., Benveniste,I., Salaun,J.P. and Durst,F. (1999) Biochem. Biophys. Res. Commun., 261, 156162.[CrossRef][ISI][Medline]
Li,H. and Poulos,T.L. (1994) Acta Crystallogr., D51, 2132.
Li,H. and Poulos,T.L. (1997) Nat. Struct. Biol., 4, 140146.[ISI][Medline]
MacKerell,A.D.,Jr et al. (1998) J. Phys. Chem. B, 102, 35863616.[CrossRef][ISI]
Mizutani,M., Ohta,D. and Sato,R. (1997) Plant Physiol., 113, 755763.
Nomura,T., Ishihara,A., Imaishi,H., Endo,T.R., Ohkawa,H. and Iwamura,H. (2002) Mol. Genet. Genomics, 267, 210217.[CrossRef][ISI][Medline]
Peterson,J.A. and Graham-Lorence,S.A. (1995) Cytochrome P450: Structure, Mechanism and Biochemistry. 2nd edn. Plenum Press, New York, pp. 151180.
Poulos,T.L., Finzel,B.C. and Howard,A.J. (1987) J. Mol. Biol., 195, 687700.[ISI][Medline]
Ravichandran,K.G., Boddupalli,S.S., Hasemann,C.A., Peterson,J.A. and Deisenhofer,J. (1993) Science, 261, 731736.[ISI][Medline]
Schalk,M. and Croteau,R. (2000) Proc. Natl Acad. Sci. USA, 97, 1194811953.
Schoch,G., Goepfert,S., Morant,M., Hehn,A., Meyer,D., Ullmann,P. and Werck-Reichhart,D. (2001) J. Biol. Chem., 276, 3656636574.
Schoenbohm,C., Martens,S., Eder,C., Forkmann,G. and Weisshaar,B. (2000) Biol. Chem., 381, 749753.[ISI][Medline]
Schuler,M.A. (1996) Crit. Rev. Plant Sci., 15, 235284.[ISI]
Schuler,M.A. and Werck-Reichhart,D. (2003) Annu. Rev. Plant Biol., 54, 629667.[CrossRef][Medline]
Szekeres,M., Németh,K., Koncz-Kálmán,Z., Mathur,J., Kauschmann,A., Altmann,T., Rédei,G.P., Nagy,F., Schell,J. and Koncz,C. (1996) Cell, 85, 171182.[ISI][Medline]
Tijet,N., Helvig,C., Pinot,F., Le Bouquin,R., Lesot,A., Durst,F., Salaun,J.P. and Benveniste,I. (1998) Biochem. J., 332, 583589.[ISI][Medline]
Urban,P., Mignotte,C., Kazmaier,M., Delorme,F. and Pompon,D. (1997) J. Biol. Chem., 272, 1917619186.
Wellesen,K., Durst,F., Pinot,F., Benveniste,I., Nettesheim,K., Wisman,E., Steiner-Lange,S., Saedler,H. and Yephremov,A. (2001) Proc. Natl Acad. Sci. USA, 98, 96949699.
Werck-Reichhart,D. (1995) Drug Metabol. Drug Interact., 12, 220243.
Werck-Reichhart,D. and Feyereisen,R. (2000) Genome Biol., 1, REVIEWS3003.[Medline]
Werck-Reichhart,D., Bak,S. and Paquette,S. (2002) In Somerville,C.R. and Meyerowitz,E.M. (eds), The Arabidopsis Book. American Society of Plant Biologists, Rockville, MD. [on-line] doi/10.1199/tab.0009; http://www.aspb.org/publications/arabidopsis/
Williams,P.A., Cosme,J., Sridhar,V., Johnson,E.F. and McRee,D. (2000) Mol. Cell, 5, 121131.[ISI][Medline]
Received January 10, 2003; revised July 31, 2003; accepted August 20, 2003.