Key substrate recognition residues in the active site of a plant
cytochrome P450, CYP73A1
Homology model guided site-directed mutagenesis
Guillaume A. Schoch
1
, Roger Attias
2
, Monique Le Ret
1
and Danie
`
le Werck-Reichhart
1
1
Department of Plant Stress Response, Institute of Plant Molecular Biology, Universite
´
Louis Pasteur, Strasbourg, France;
2
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite
´
Paris V, 45 Paris, France
CYP73 enzymes are highly conserved cytochromes P450 in
plant species that catalyse the regiospecific 4-hydroxylation
of cinnamic acid to form precursors of lignin and many other
phenolic compounds. A CYP73A1 homology model based
on P450 experimentally solved structures was used to iden-
tify active site residues likely to govern substrate binding and
regio-specific catalysis. The functional significance of these
residues was assessed using site-directed mutagenesis. Active
site modelling predicted that N302 and I371 form a hydro-
gen bond and hydrophobic contacts with the anionic site or

that the cinnamate 4-hydroxylase (C4H) activity proceeds
with a perfect coupling of oxygen consumption and
reducing equivalents to produce hydroxylated substrates
[3]. CYP73A1 provides a good model for determining the
residues that control catalytic efficiency and optimal
substrate positioning in a typical plant P450 enzyme
contributing to a high throughput anabolic pathway.
CYP73A1 is one of the most extensively studied plant
P450 enzymes. It has a quite high substrate specificity but
can accommodate a diverse array of compounds, as far as
they are structural analogues of the natural substrate.
Structural requirements for such analogues include a planar,
aromatic structure, a small size of about two adjacent
aromatic rings, and an anionic site opposite (i.e. at about
8.5 A
˚
) to the position of oxidative attack [7,8]. A recent site-
directed mutagenesis study that investigated the role of
unusual residues in the most conserved regions involved in
haem binding and oxygen activation [9], suggested that
some are likely to contribute to the optimal coupling of the
C4H reaction. The protein residues that govern substrate
recognition and orientation have not yet been identified.
In order to obtain information on the orientation and
positioning of the substrates in the active site, we have
recently engineered a stable and water-soluble form of
CYP73A1 that is suitable for
1
H-NMR paramagnetic
relaxation experiments [10]. The results of the NMR

Experimental procedures
Chemicals
Trans-cinnamic acid (CA), trans-cinnamaldehyde, indole-
3-acetic acid (IAA), indole-2-carboxylic acid (I2C), indole-
3-carboxylic acid (I3C), 7-methoxycoumarin (7MC),
2-naphthoic acid (NA), phenylpyruvic acid, NADPH and
umbelliferone were from Sigma-Aldrich (l’Isle d’Abeau
Chesnes, France). trans-Cinnamylic alcohol and 6-hydroxy-
2-naphthoic acid were from Lancaster Synthesis (Stras-
bourg, France).
L
(–)-Phenylalanine and naphthalene-1-acetic
acid were from Merck (Schuchardt, Germany). 2-Amino-
quinoline and 2-phenoxyacetamidine were from Maybridge
(Tintagel, UK), 5-hydroxy-2-indolecarboxylic acid was
from Acros Organics (Noisy-Le-Grand, France), trans-
[3-
14
C]cinnamate was from Isotopchim (Ganagobie,
France). 4-Propynyl-oxybenzoic acid was a gift from
W. Alworth (Tulane University, New Orleans).
Mutagenesis
The modified CYP73A1 cDNAs were generated using
QuickChange
TM
Site-Directed Mutagenesis (Stratagene)
using as a template the double-stranded wild-type
CYP73A1 cDNA from Helianthus tuberosus (GenBank
Z17369) subcloned as an EcoRI–BamHI fragment into the
shuttle vector pYeDP60 [12] and the primers listed in

and 72 °C, 22 min) followed by 10 min extension at 72 °C.
Parental methylated DNA was selectively digested with
DpnI before transformation of Escherichia coli. The inserts
of the selected neosynthetized vectors were fully sequenced.
As neosynthetized DNA is not a template for the reaction,
the amplification is linear, which is expected to keep the
error frequency low in the final PCR product. Two
problems were, however, encountered in our experiments:
additional mutations around the site of mutagenesis and a
large proportion of wild-type vectors were frequently
obtained. As controls showed that the parental DNA was
digested, this was attributed to poor primer synthesis or
correcting properties of the polymerase.
Yeast expression and microsome preparation
The pYeDP60 vector [12] and the modified strain of
Saccharomyces cerevisae W(R) over-expressing its own
NADPH-P450 reductase were used for the expression of
the constructs [13]. Yeast transformation was performed as
described in [14], growth and induction were based on the
high density procedure described in [15]. To achieve optimal
expression, a yeast colony grown on an SGI plate was
tooth-picked into 50 mL SGI and grown for 18 h at 30 °C
to a density of 6 · 10
7
cellsÆmL
)1
. This preculture was
diluted in YPGE to a density of 2 · 10
5
cellsÆmL

DKR 5¢-GAAGTTAAAGATACAATGATTCAGCTC 5¢-GAGCTGAATCATTGTAACTTTAACTTC-3¢ 48
N302D 5¢-CATTGTTGAAGACATCAATGTTG-3¢ 5¢-CAACATTGATGTCTTCAACAATG-3¢ 43
N302F 5¢-CTTTACATTGTTGAATTCATCAATGTTGCAGC-3¢ 5¢-GCTGCAACATTGATGAATTCAACAATGTAAAG-3¢ 43
I303A 5¢-CATTGTTGAAAACGCTAATGTTGCAG-3¢ 5¢-CTGCAACATTAGCGTTTTCAACAATG-3¢ 52
R366M 5¢-CAAGGAAACCCTCATGCTCCGTATG-3¢ 5¢-CATACGGAGCATGAGGGTTTCCTTG-3¢ 55
R368K 5¢-CCCTCCGTCTCGAAATGGCGATCCG-3¢ 5¢-CGGGATCGCCATTTCGAGACGGAGGG-3¢ 50
R368F 5¢-CCCTCCGTCTCTTTATGGCGATCCG-3¢ 5¢-CGGGATCGCCATAAAGAGACGGAGGG-3¢ 50
I371F 5¢-TCCGTATGGCGTTCCCGCTTCTAGTC-3¢ 5¢-GACTAGAAGCGGGAACGCCATACGGA-3¢ 58
I371A 5¢-TCCGTATGGCGGCTCCGCTTCTAGTC-3¢ 5¢-GACTAGAAGCGGAGCCGCCATACGGA-3¢ 58
I371K 5¢-TCCGTATGGCGAAACCGCTTCTAGTC-3¢ 5¢-GACTAGAAGCGGTTTCGCCATACGGA-3¢ 58
K484M 5¢-GATACCGATGAGATGGGTGGGCAGTTTAG-3¢ 5¢-CTAAACTGCCCACCCATCTCATCGGTATC-3¢ 58
Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3685
dissociation constants of enzyme–ligand complexes were
evaluated from Type I ligand binding spectra using the
e
peak-trough
¼ 125Æm
M
)1
Æcm
)1
[7]. Integrity of the enzyme
was checked at the end of each titration experiment by
recording a difference spectrum of the CO-reduced protein.
Cytochrome c reductase activity of the NADPH-cyto-
chrome P450 reductase was assayed as in [17]. Trans-CA
hydroxylation was assayed using radiolabelled trans-
[3-
14
C]CA and TLC analysis of the metabolites [18]. For

times of I3C and its oxygenated product were 13 and
6.5 min, respectively. Products of I2C incubation were
collected, evaporated and submitted to MS analysis on a
BioQ triple quadrupole (Micromass).
Phenylalanine, which is insoluble at pH 7.4, was dissolved
in sodium borate 100 m
M
pH 8.3. Phenylalanine and
2-phenoxyacetamidine hydroxylations were assayed by
HPLC under similar conditions as I2C and I3C, excepted
for phenylalanine mobile phase (isocratic 5% acetonitrile,
7.5 m
M
(NH
4
)
2
PO
4
,7.5m
M
HCl). NA hydroxylation was
assayed by fluorometry [7] in 2 mL 100 m
M
sodium
phosphate pH 7.4 containing 0.2, 0.5 or 1 mg yeast micro-
somal protein, 600 l
M
NADPH, and 100 l
M

by this method (about 35 000 constraints were kept for the
present application). The input data to the modified
DYANA
program are then no longer NMR constraints, but
geometrical distances and torsions derived statistically from
the templates.
DYANA
minimization includes Van de Waals’
interaction calculations, and proposes its best solution from
a starting conformation.
Structures were analysed by using the
PROCHECK
package
and Accelrys
INSIGHTII
. Model minimization was further
refined with the functionalities of Accelrys
DISCOVER
3
(version 97.0, Force Field CVFF and ESFF when including
the haem iron atom). At this stage, electrostatic interactions
are included in the minimization process. At each of the
modelling steps, models are selected on the basis of quality
scores supplied by the related program (f factor in
DYANA
,
or
PROCHECK
G-factors scoring ideally above )0.5 for
instance).

The atoms of the side chains showing identical spatial
location when superimposing each set of residues were
considered as conserved atoms. They were identified and
added to the list of the block backbone conserved atoms.
These side chain atoms also provide the resulting rotamer
value for the related target residue. Other rotamers, for
residues with no conserved side chain atom, were attributed
by using a rotamer library [24].
From the three-dimensional coordinates of the common
structural blocks, we derived a set of geometrical constraints
(mean distances between two atoms, mean Phi and Psi
values), and their standard deviations. The distance cutoff
between two atoms was set to 5 A
˚
, except for interblock CB
atoms where no cutoff was given in order to reflect the more
flexible relative location of the blocks. These constraints
constitute, within a tolerance interval, the spatial informa-
tion that was used to build the model. The
DYANA
program
was then used to calculate initial random coordinates of the
target protein and performed minimization under this set of
distance and dihedral constraints [25]. The loops between
the blocks were built with no constraints. From each model,
Phi and Psi additional constraints for nonconserved residues
were derived in order to restrain them in an allowed region
of the Ramachandran region.
DYANA
was then rerun and

blocks were used to assign three-dimensional coordinates to
the corresponding blocks in CYP73A1. The resulting model
of the CYP73A1 core structure and active site region is
represented in Fig. 2. The advantage of this approach is that
it merges structural information from several known
structures into the target protein rather than producing a
model that is based on a single structure. All techniques are
limited by the prediction of the protein alignments, but
integration of information from multiple structures has
some chances to be better when, as in our case, protein
identities are very low.
The 6–8 A
˚
distances between the substrate protons and
the haem iron were recently deduced from
1
H paramagnetic
relaxation experiments [10] indicate that CA initially binds
roughly parallel to the haem in the oxidized CYP73A1. The
carboxylic function, which can be replaced by other anionic
groups, was previously shown to be an essential determinant
of substrate docking in the active site [7,8]. An ionic or
hydrogen bond is likely to anchor CA to a cationic or
hydrophilic residue of the protein. These data suggest that a
set of residues within 5–9 A
˚
above haem iron could be
considered putative active site contacts and tested by site-
directed mutagenesis. A search of the model for hydrophilic
residues likely to form a hydrogen bond with the substrate

Fig. 1. Predicted location of the conserved structural blocks and SRSs
on the primary sequence of CYP73A1. Sequence alignments of
CYP73A1 with the common structural blocks of four bacterial crystal
structures (P450
BM3
, P450
CAM
, P450
TERP
,andP450
eryF
)predicted
some of the substrate recognition sites regions. SRS locations were
corroborated on the basis of a multiple alignment with the four bac-
terial enzymes also including some members of the CYP2 and CYP73
families. CYP73A1 putative SRSs determined on the basis of this
alignment are underlined (numbered 1–6 from N to C terminal) and
residues selected for directed mutagenesis are indicated by stars. The
region interblocks in CYP73A1 are displayed in grey. For the bacterial
sequences only the common structural blocks are represented, the
identity between sequences is shaded in black, similarity is shaded in
grey (threshold of 70%).
Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3687
bulk, except in the case of hydrophobic contacts for which
the influence of side-chain size was investigated. The
consecutive residues K248 and R249 were modified simul-
taneously to avoid charge compensation. As the desired
double mutations were not obtained, we analysed the triple
mutants D247E/K248T/R249M (DKR) and K248T/
R249M/I371K (KRI).

1/2
) of the wild-type
CYP73A1 was around 3 h. In the presence of a higher
sodium dithionite concentration (4.5 mgÆmL
)1
), the t
1/2
of
CYP73A1 was 45 min when the buffer contained 3%
glycerol, and 60 min with 30% glycerol. Stability tests were
performed at 4.5 mgÆmL
)1
dithionite, using different con-
centrations of glycerol depending on the stability of each
mutant (Table 2). The results identified three classes of
mutants. The first group consisted of N302D, I371F and
I371K, that had a stability at least equal to that of the wild-
type. The second group included K484M, with a stability
that was slightly decreased compared to the wild-type, and
R103M, N302F and I371A that displayed a more pro-
nounced decrease with a t
1/2
shift from 45 to approximately
15 min. The third group, included all other mutants in
particular R366M and R101M, which demonstrated a
drastic loss in protein stability. The R366M, R368F/K,
R101M, R103M/E, DRK and KRI modifications resulted
in a very significant disruption of the tertiary structure of
the CYP73A1 protein.
Effect of mutations on cinnamic acid recognition

M
pH 7.4 containing 3% glycerol. (2) Low stability mutants were tested in buffer containing 30% glycerol to underline the differences between them. C4H activity was
measured using a concentration of cinnamate (150 l
M
) expected to be saturating for most of the mutants. The binding constants were calculated from the amplitude of the type I difference spectra induced by
increasing concentrations of substrate, e
type I
being the molar absorption coefficient of the saturated P450-substrate complex (DA
max
/P450 concentration) and K
s
the dissociation constant. Expression and
activity values are relative to the wild-type (100%): P450 expression, 847 pmolÆmg
)1
microsomal protein; C4H activity, 287 pkatÆmg
)1
; cytochrome c reductase activity, 1520 pkatÆmg
)1
. Cytochrome c
reductase activity is used as a control for protein induced expression and integrity. Values ± SD are the mean of three or more experiments. n.m. not measurable.
Hydrophobic and hydrogen bonding residues Positively charged residues
I helix (SRS 4) Loop 3 (SRS 5) B helix (SRS 1) (SRS 3) K helix (SRS 5) (SRS 6)
Wild-type N302F N302D I303A I371F I371A I371K Wild-type R101M R103M R103E DKR KRI R366M R368K R368F K484M
Yeast
expression
level (%)
100 ± 4.8 30 ± 0.3 71 ± 7.2 96 ± 5.1 95 ± 9.9 93 ± 2.8 105 ± 2.4 100 ± 4.8 11.2 ± 0.7 60 ± 3.5 78 ± 1.7 7.8 ± 0.2 11.6 ± 0.8 <5 81 ± 3.3 54 ± 4.3 89 ± 1.4
Initial P420
(%)
– 15 – <5 – – – – 50 – 5 65 <5 >80 – – –

had no significant impact on the binding of CA; however, it
did result in a 45% decrease in catalytic activity. All other
modifications of positively charged amino acids adversely
affected expression and/or stability of the enzyme but had a
comparatively minor affect on substrate recognition and
metabolism. Exceptions included R366M, R101M and the
triple mutations for which drastic decreases in stable haem
protein were paralleled by dramatic losses in activity.
Despite the loss of activity and structural integrity, the
DKR mutation rather unexpectedly seemed to retain an
intact affinity for substrate binding.
Modifications of N302 and I371 resulted in limited or no
apparent perturbation of protein folding and stability but
led to dramatic decreases in CA binding and hydroxylation.
N302 is likely to provide a hydrogen bonding side chain for
anchoring the carboxylate of CA. The conversion of aspa-
ragine into negatively charged aspartic acid (N302D)
resulted in a drastic effect on substrate binding affinity.
Whereas replacement with a bulky hydrophobic residue
(N302F) compromised overall protein structure and cata-
lysis.
I371 is predicted to form a van der Waals’ contact with
the aromatic ring of CA. In the I371 mutants, I371A opens
more space in the active site and thus should allow for
increased substrate mobility. Conversely, I371F and I371K
should create a steric hindrance to the binding of the
substrate above the haem iron. As expected, the I371A
mutation substantially decreases CA affinity and the ability
to desolvate the active site. Around 10% of the catalytic
activity is conserved, which would be in agreement with the

s
¼ 12 l
M
), 2-aminoquinoline (K
s
¼ 17 l
M
) and indole-
3-carboxylic acid (the natural auxin, K
s
¼ 18 l
M
). These
compounds are ordered from gain to loss of binding to the
mutant proteins in Table 3.
As shown in Table 3, the analogues investigated were
better ligands for the mutants than the physiological
substrate CA. Relative to wild-type CYP73A1, the binding
efficiency for CA decreases 10-fold in the mutant I371K,
50-fold in I371F and 100-fold in N302D. In contrast,
increases in binding efficiency are observed for a few ligands
after modification of the protein. The most notable increases
are 15-fold for N302D with phenylalanine, 12-fold for
I371K with 2-phenoxyacetamidine, and 10-fold for I371F
with phenylalanine or cinnamylic alcohol.
The I371F modification is likely to block access to the
active centre for most of the potential substrates. Only
compounds with increased side chain flexibility or reduced
bulkiness in the CA ring region are expected to have
increased binding efficiencies compared to CA. This is

NA was previously shown to be the best structural mimic
and alternate substrate for wild-type CYP73A1 [7]. NA was
metabolized by all mutants with an efficiency very compar-
able to that observed with CA. This suggests that both
compounds have a very similar positioning in the active site
and validates use of NA for fluorometric quantification of
the enzyme activity [7]. Metabolism of I2C, I3C and 7MC
does not parallel that of CA in the different mutants. For
example the I371A and I371K mutations have less influence
on demethylation of 7MC than on CA hydroxylation. Also
noteworthy is the opposite effect of several amino acid
substitutions on I3C and I2C hydroxylations. Most muta-
tions have less impact on I2C than on I3C and CA
metabolism, probably due to the symmetry axis of I2C and
to the possible attack on two different carbon atoms.
3690 G. A. Schoch et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Unexpectedly, the K484M substitution, which results in
close to 50% loss in C4H activity, does not affect I2C
hydroxylation. As initial cinnamate binding is not influ-
enced by this mutation (Table 2) and binding kinetics are
first-order (indicating a single binding-site), this suggests
that K484 does not directly affect catalysis but might have a
selective role in substrate position adjustment during the
catalytic cycle.
Modified regiospecificity of indole-2-carboxylic acid
hydroxylation
I2C metabolism by wild-type CYP73A1 was previously
shown to result in the formation of two products that were
not further characterized [7]. On the basis of its HPLC
retention time, UV spectrum, and monoisotopic mass, the

s
ratio calculated for each complex. The
values listed are relative to the wild-type for each ligand. Standard deviations (not shown) are less than 12% of these values.
Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3691
molecule in the active site that is reflected by a low e
type I
(Table 3). Taken together, the data support a role for N302
in controlling of substrate orientation in the active site.
The I371 mutations to K and A have opposite effects.
The I371K mutation increases the preferential attack at the
putative 6-position, most likely by increasing steric hin-
drance near C5 of the indole ring. In contrast, the I371A
mutation appears to remove the steric constraint existing in
the wild-type CYP73A1 and favours a P2/P1 ratio closer
to 1. The observed effects of both of these mutations
support the assumption of a direct contact of I371 with the
aromatic ring of I2C or CA.
Discussion
The computational homology modelling strategy des-
cribed by Jean et al. [21] allows a reasonable prediction
of the most conserved P450 substructures, although
hypervariable regions cannot be predicted. Our present
model was based on four crystallized bacterial enzymes
(Fig. 2) and seems to correctly predict several residues
forming contacts with CA.
The model predicts that N302, which resides in the I helix
and SRS 4, is likely to form a hydrogen bond with the
carboxylate of the substrate. Mutations of this residue lead to
a dramatic loss in CA binding efficiency (10-fold for the
N302F and 100-fold for the N302D substitution) together

haem, while interaction with the reductase and electron
transfer should involve residues on the proximal side of the
protein [31]. The unchanged I2C hydroxylase activity in
K484M when compared to that of the wild-type confirms
that the mutant is not impaired in electron transfer. Thus,
K484 must exert some control on CA/NA positioning or
product release during the catalytic cycle. Although the
K484 effect might be indirect and the interaction with the
carboxylate of CA might occur via a molecule of solvent, it
can also be postulated that the reduction of the protein or
binding of oxygen results in a conformational change of the
Fig. 3. Analysis of the products of I2C hydroxylation. Upper panel:
HPLC analysis of the products of the metabolism of 10 nmol I2C by
30 pmol recombinant CYP73A1 in 60 min and in a 100 lL assay.
Absorbance was monitored at 290 nm. Lower panel: UV spectra
corresponding to the centre of the peaks. P1 and P2 collected after
90 min incubation of 120 nmol of I2C were analysed by negative ESI-
MS. Monoisotopic mass of both compounds was 176 Da. P1 retention
time and UV spectrum was identical to that of commercial 5-hydroxy-
2-indolecarboxylic acid.
Table 4. Metabolism of alternate substrates by mutant CYP73A1s. Activities are expressed relative to wild-type CYP73A1. 100% activity is
287 pkatÆmg
)1
microsomal protein for CA, 311 pkatÆmg
)1
for NA, 38.8 pkat mg
)1
for I2C, 20.4 pkat mg
)1
for I3C, 6.6 pkat mg

at the 4 position or on the 3–4 bond, would be needed for
efficient and regiospecific catalysis. The K484M mutation
has no impact on I2C metabolism or the regioselectivity of
attack. This observation is compatible with a role of K484 in
CA reorientation as the slightly smaller size and different
shape of I2C compared to that of CA might prevent
interaction between its anionic site and K484.
N302 and I371 align with residues that have been
shown to confer substrate specificity or regioselectivity to
many other plant or mammalian P450 enzymes. Residues
corresponding to I371 govern the regiospecificity of the
hydroxylation of 4S-limonene in CYP71D18 from spear-
mint and CYP71D15 from peppermint for the synthesis
of carvone and menthol, respectively [28]. In the
mammalian CYP2B family, residues 294 and 363 are
equivalent as N302 and I371, respectively. The CYP2B
mutations were shown to affect steroid regioselectivity.
At position 363, a CYP2B1 mutant (V363L) exhibited a
twofold decrease in androgen activity [35], whereas in
CYP2B11 the reverse mutant shows a fivefold increase in
androgen activity [36]. The same residue was identified as
a determinant of substrate specificity in CYP2B2 [37],
CYP2B5 [38] and CYP2B6 [39]. Likewise, residue 294
was shown to play a key role in androgen metabolism by
CYP2B1 [40] and CYP2B4 [38]. A similar affect on
catalysis by these residue positions has been reported for
other mammalian enzymes. For example in CYP2A5,
mutation of M365, the equivalent of I371, decreased the
metabolism of aflatoxin B1 [41], while modification of
the corresponding residue (A370) in human CYP3A4

new structure confirmed the conservation of the P450
spatial organization in eukaryotic microsomal enzymes. The
position of SRS 4 that is located in the centre of the I helix,
which includes N302 in CYP73A1, was highly conserved
relative to the haem. However, significant local changes
were detected, particularly in all other SRSs. For example,
SRS 5, facing the I helix, shows a double bend due to two
proline residues (P360 and P364). The resulting topology
orients three leucine side chains toward the active site (L358,
L359 and L363). In P450
CAM
[46] and P450
TERP
[47], SRS 5
is a b-strand partially involved in b-sheet formation with
SRS 6. In P450
BM3
[44], the first bend found in CYP2C5 is
present and the C-terminal part of SRS 5 is a b-strand not
involved in a b-sheet with SRS 6. The alignment of SRS 5
of CYP2C5 and the whole CYP2B family with those of
CYP73A1 and CYP71Ds is not ambiguous. The two
prolines and the adjacent positive charge (H365) that bind
the haem propionate in CYP2C5 are conserved. This
suggests that the double bend structure is present and
confirms I371 as a central residue of SRS 5 in CYP73A1. If
the position of the SRS relative to the haem is conserved, the
phenyl side chain in the I371F mutant should stack over the
haem, which would explain the complete impairment of
substrate binding and the increased stability of the mutant

from the haem iron and
3.5 A
˚
from the substrate. K484 is still too far away to form a
direct contact with the cinnamate.
In conclusion, a combination of homology modelling
and site-directed mutagenesis of CYP73A1 has identified
N302 and I371 as key determinants of substrate binding
and orientation for catalysis. K484 is not involved in
initial substrate binding, but seems to play a significant
role in catalysis, possibly by contributing to substrate
reorientation during the catalytic cycle. Modification
of active site residues improved affinity for substrate
Ó FEBS 2003 Key residues for substrate recognition in CYP73A1 (Eur. J. Biochem. 270) 3693
analogues, but correct positioning allowing for a gain of
function could not be achieved. Indole 2-carboxylic acid,
which is regiospecifically attacked at the 5 and 6
positions, is a very useful probe for investigating the
topology of the CYP73A1 active site.
Acknowledgments
We thank P. Ullmann for help and support, M. Bergdoll for helpful
discussion, D. Little and K. Griffin for critical readings of the
manuscript. The W(R) and WAT11 yeast strains and the pYeDP60
expression vector were kindly provided by Drs D. Pompon and
P. Urban (CNRS, Gif-sur-Yvette). This work was supported by the
CNRS Program Chimie-Physique du Vivant, and a fellowship from the
French Ministry of Research to G.A.S.
References
1. Weisshaar, B. & Jenkins, G.I. (1998) Phenylpropanoid biosyn-
thesis and its regulation. Curr. Opin. Plant Biol. 1, 251–257.

metabolites in the phenylpropanoid pathway. Plant Physiol. 118,
209–218.
9. Schalk, M., Nedelkina, M., Schoch, G., Batard, Y. & Werck-
Reichhart, D. (1999) Role of unusual amino acid residues in the
proximal and distal heme regions of a plant P450, CYP73A1.
Biochemistry 38, 6093–6103.
10. Schoch, G.A., Attias, R., Belghazi, M., Dansette, P.M. & Werck-
Reichhart, D. (2003) Engineering of a water-soluble plant cyto-
chrome P450, CYP73A1, and NMR based orientation of natural
and alternate substrates in the active site. Plant Physiol., in press.
11. Williams, P.A., Cosme, J., Sridhar, V., Johnson, E. & McRee,
D.E. (2000) Mammalian microsomal cytochrome P450 mono-
oxygenase: structural adaptations for membrane binding and
functional diversity. Mol. Cell 5, 121–131.
12. Urban, P., Cullin, C. & Pompon, D. (1990) Maximizing the
expression of mammalian cytochrome P-450 monooxygenase
activities in yeast cells. Biochimie 72, 463–472.
13. Truan, G., Cullin, C., Reisdorf, P., Urban, P. & Pompon, D.
(1993) Enhanced in vivo monooxygenase activities of mammalian
P450s in engineered yeast cells producing high levels of
NADPH-P450 reductase and human cytochrome b
5
. Gene 125,
49–55.
14. Urban, P., Werck-Reichhart, D., Teutsch, H.G., Durst, F., Reg-
nier, S., Kazmaier, M. & Pompon, D. (1994) Characterization of
recombinant plant cinnamate 4-hydroxylase produced in yeast.
Kinetic and spectral properties of the major plant P450 of the
phenylpropanoid pathway. Eur. J. Biochem. 222, 843–850.
15. Pompon,D.,Louerat,B.,Bronine,A.&Urban,P.(1996)Yeast

Two cytochrome P-450 isoforms catalysing O-de-ethylation of
ethoxycoumarin and ethoxyresorufin in higher plants. Biochem.
J. 270, 729–735.
21.Jean,P.,Pothier,J.,Dansette,P.M.,Mansuy,D.&Viari,A.
(1997) Automated multiple analysis of protein structures: appli-
cation to homology modeling of cytochromes P450. Proteins 28,
388–404.
22. Minoletti, C. (1998) PhD Thesis. University Paris VI Rene
´
Descartes, Paris, France.
23. Patard, L., Stoven, V., Gharib, B., Bontems, F., Lallemand, J.Y.
& De Reggi, M. (1996) What function for human lithostathine?:
structural investigations by three-dimensional structure modeling
and high-resolution NMR spectroscopy. Protein Eng. 9, 949–957.
24. Dunbrack, R.L. Jr & Karplus, M. (1993) Backbone-dependent
rotamer library for proteins. Application to side-chain prediction.
J. Mol. Biol. 230, 543–574.
25.Guntert,P.,Mumenthaler,C.&Wuthrich,K.(1997)Torsion
angle dynamics for NMR structure calculation with the new
program DYANA. J. Mol. Biol. 273, 283–298.
26. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton,
J.M. (1993) PROCHECK: a program to check the streochemical
quality of protein structures. J. Appl. Cryst. 26, 283–291.
27. Gotoh, O. (1992) Substrate recognition sites in cytochrome P450
family 2 (CYP2) proteins inferred from comparative analyses of
amino acid and coding nucleotide sequences. J. Biol. Chem. 267,
83–90.
28. Schalk, M. & Croteau, R. (2000) A single amino acid substitution
(F363I) converts the regiochemistry of the spearmint (–)-limonene
hydroxylase from a C6- to a C3-hydroxylase. Proc.NatlAcad.Sci.

K.M.,Burnett,V.L.,He,Y.A.,Kaminsky,L.S.&Halpert,J.R.
(1994) Site-directed mutagenesis of putative substrate recognition
sites in cytochrome P450 2B11: importance of amino acid residues
114, 290, and 363 for substrate specificity. Mol. Pharmacol. 46,
338–345.
37. Strobel, S.M. & Halpert, J.R. (1997) Reassessment of cytochrome
P450 2B2: catalytic specificity and identification of four active site
residues. Biochemistry 36, 11697–11706.
38. Szklarz,G.D.,He,Y.Q.,Kedzie,K.M.,Halpert,J.R.&Burnett,
V.L. (1996) Elucidation of amino acid residues critical for unique
activities of rabbit cytochrome P450 2B5 using hybrid enzymes
and reciprocal site-directed mutagenesis with rabbit cytochrome
P450 2B4. Arch. Biochem. Biophys. 327, 308–318.
39. Domanski, T.L., Schultz, K.M., Roussel, F., Stevens, J.C. &
Halpert, J.R. (1999) Structure–function analysis of human cyto-
chrome P-450 2B6 using a novel substrate, site-directed muta-
genesis, and molecular modeling. J. Pharmacol. Exp. Ther. 290,
1141–1147.
40. Domanski, T.L., He, Y.Q., Scott, E.E., Wang, Q. & Halpert, J.R.
(2001) The role of cytochrome 2B1 substrate recognition site
residues 115, 294, 297, 298, and 362 in the oxidation of steroids and
7-alkoxycoumarins. Arch. Biochem. Biophys. 394, 21–28.
41. Pelkonen, P., Lang, M.A., Negishi, M., Wild, C.P. & Juvonen,
R.O. (1997) Interaction of aflatoxin B1 with cytochrome P450 2A5
and its mutants: correlation with metabolic activation and toxicity.
Chem. Res. Toxicol. 10, 85–90.
42. He, Y.A., He, Y.Q., Szklarz, G.D. & Halpert, J.R. (1997) Iden-
tification of three key residues in substrate recognition site 5
of human cytochrome P450 3A4 by cassette and site-directed
mutagenesis. Biochemistry 36, 8831–8839.