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OCR for page 81
Colloquium
Diversification of furanocoumarin-metabolizing
cytochrome P450 monooxygenases in two papilionicis:
Specificity and substrate encounter rate
Weimin Li*, Mary A. Schulert, and May R. Berenbaum*'
Departments of *Entomology and tCell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801
Diversification of cytochrome P450 monoaxygenases (P450s) is
thought to result from antagonistic interactions between plants
and their herbivorous enemies. However, little direct evidence
demonstrates the relationship between selection by plant toxins
and adaptive changes in herbivore P450s. Here we show that the
furanocoumarin-metabolic activity of CYP6B proteins in two spe-
cies of swallowtail caterpillars is associated with the probability of
encountering host plant furanocoumarins. Catalytic activity was
compared in two closely related CYP6B4 and CYP6B17 groups in the
polyphagous congeners Papilio glaucus and Papilio canadensis.
Generally, P450s from P. glaucus, which feeds occasionally on
furanocoumarin-containing host plants, display higher activities
against furanocoumarins than those from P. canadensis, which
normally does not encounter furanocoumarins. These P450s in turn
catalyze a larger range of furanocoumarins at lower efficiency than
CYP6B1, a P450 from Papi/io polyxenes, which feeds exclusively on
furanocoumarin-containing host plants. Reconstruction of the an-
cestral CYP6B sequences using maximum likelihood predictions
and comparisons of the sequence and geometry of their active sites
to those of contemporary CYP6B proteins indicate that host plant
diversity is directly related to P450 activity and inversely related to
substrate specificity. These predictions suggest that, along the
lineage leading to Papilio P450s, the ancestral, highly versatile
CYP6B protein presumed to exist in a polyphagous species evolved
through time into a more efficient and specialized CYP6B1-like
protein in Papilio species with continual exposure to furanocou-
marins. Further diversification of Papilio CYP6Bs has likely involved
interspersed events of positive selection in oligophagous species
and relaxation of functional constraints in polyphagous species.
insects I metabolism I molecular modeling
Cytochrome P450 monooxygenases (P450s) comprise a vast
superfamily of heme-thiolate enzymes that catalyze the
NADPH-associated reductive cleavage of oxygen to produce a
fictionalized product and water (1~. The genes encoding these
enzymes constitute one of the lamest known gene superfamilies
(http://drnelson.utmem.edu/CytochromeP450.html), with the
enormous proliferation reflecting the functional versatility of
their encoded proteins. Studies in a wide variety of organisms
have demonstrated that P450-catalyzed reactions are important
for detoxification of exogenous compounds, such as drugs, toxic
pollutants, pesticides, and plant allelochemicals, as well as
biosynthesis of endogenous agents, such as steroid hormones,
pheromones, and defense compounds (2-5~.
It has been suggested that, although the earliest P450s in
eukaryotes were important in the metabolism of endogenous
substrates, convolution between plants and herbivorous animals,
including insects, expedited the diversification of P450 families
(6, 74. The earliest eukaryotic P450s in both plants and animals
used reactive oxygen to metabolize endogenous compounds,
www.pnas.org/cgi/doi/ ~ 0. ~ 073/pnas. ~ 934643 ~ 00
such as steroids and fatty acids. Subsequent reciprocal adaptive
selection between plants and herbivorous animals was associated
with the rapid diversification of P450s initiating 400 million years
ago, concomitant with the colonization of terrestrial habitats by
plants and animals. Plants have used P450s to produce defense
compounds (allelochemicals), and herbivorous animals, includ-
ing insects, have used P450s to metabolize the toxins produced
by plants. Multiple duplication and divergence events are
thought to have allowed xenobiotic-metabolizing P450s, such as
CYP2 and CYP3 in mammals and CYP6 in insects, to diversify
and acquire new functions. Insect genome projects have revealed
tremendous diversity in putative xenobiotic-metabolizing P450
families, with approximately half of the 90 P450s in the Dro-
sophila melanogaster genome belonging to families CYP6 and
CYP4 (8~. In the evolution of these large gene families, Hughes
and Nei (9) and Ota and Nei (10) have proposed that duplication
events may be followed by a winnowing process whereby some
duplicate genes "die out" because of accumulation of deleterious
mutations. This "evolution by the birth-and-death process"
allows the number of functional genes within a family to remain
stable. The birth-and-death model may be particularly applicable
to the diversification of P450s in herbivorous insects, which,
during host shifts, encounter different selective forces associated
with the biochemical defense profiles of their host plants.
COMB family genes and proteins have been characterized in
two groups of lepidopterans: the Helicoverpa/Heliothis complex
and Papilio species (11~. Within the genus Papilio, proliferation
of CIP6B genes has occurred within the context of dietary
furanocoumanns, a class of secondary compounds that confer
protection against herbivores, because, on activation by UV
light, they bind covalently to DNA and protein (12, 13~. Furano-
coumarins occur in two structural configurations, linear and
angular, in over a dozen plant families, and are most widely
distributed and diverse in Rutaceae and Apiaceae, the preferred
hosts for most Papilio species (14~.
Despite the toxicity of furanocoumarins, the oligophagous
Papilio polyxenes specifically feeds on furanocoumarin-
containing Apiaceae (11~. Transcripts of at least one gene,
CYP6B1, are expressed at elevated levels in response to supple-
mental furanocoumarins (15~. The CYP6B1 protein encoded by
this gene displays very high activity against linear furanocou-
marins and considerably less activity against angular furanocou-
marins. Surprisingly, two closely related polyphagous Papilio
This paper results from the Arthur M. Sackler Colloquium of the National Academy of
Sciences, "Chemical Communication in a Post-Genomic Worict," held January 17-19, 2003,
at the Arnold and Mabel Beckman Center of the National Academies of Science and
Engineering in Irvine, CA.
Abbreviation: P450s, cytochrome P450 monooxygenases.
tTo whom correspondence should be addressed. E-mail: maybe~uiuc.ectu.
Hi 2003 by The National Academy of Sciences of the USA
PNAS 1 November 25, 2003 1 vol. 100 1 suppl. 2 1 14593-14598
OCR for page 82
Table 1. Specific activities of CYP6B proteins coexpressed with house fly NADPH P450 reductase in baculovirus expression system
Specific activity (nmol/min/nmol P450),*
means+SD
Angelicin Trioxsalen
ib~ ~
P450 CO-di~
>to
Psoralen Xanthotoxin
To
of
Berga,p,ten
Q
To
CYP6B4 (Pot) 450 1.906 ~ 0.180a 1.412 ~ o.o90a 2.208 ~ O.llSa 3.214 ~ 0.1743 3.541 :t 0.126a
CYP6B17 (Pg) 450 0.060 ~ 0.023C 0.557 ~ 0.0816 0.381 ~ 0.150b 0.800 ~ 0.251d 1.122 ~ 0.141d
(97%~) (61%~) (83%) (75%~) (68%~1)
CYP6B21 (Pg) 450 0.518~0.130b 0.773:~0.170b 1.80010.2111 2.547~0.087b 2.150~0.266b
(73%~) (45%) (18%~) (21%) (39%~1)
CYP6B25 (Pc) 450/420 0.372 ~ 0.21 ob o. 179 :t: 0.1 god 0.627 ~ 0.346b 1.212 ~ 0.1 60C 1.486 ~ 0.1 24c
(80%J') (87%~) (72%~) (62%~) (58%~)
CYP6B26 (Pc) 420 ND ND ND ND ND
(100%~) (100%~1) (100%~) (100%J<) (100%~)
CYP6B1 ' 450 0.640 . 2.560 6.980 _
*Specific activities for each furanocoumarin were compared, and significant differences are represented with suDerscriDt letters (P < 0.05) (ANOVA test).
tCYP6B1 activities were measured at different ratio of MOls at 4:1 of P450 vs. P450-NADPH reductase recombinant viruses (28).
tNot tested.
species, Papilio glaucus, which occasionally encounters furano-
coumarins, and Papilio canadensis, which is unlikely to encounter
furanocoumarins because of the absence of furanocoumarins in
its available host plants, also have inducible metabolisms of
furanocoumarins (16~. Sixteen highly conserved genes (92-99%
protein identity) belonging to two groups, designated the
CYP6B4 and CYP6B17 groups, have been isolated from these
two species (16, 17~. Although all of these P450 transcripts are
inducible by furanocoumarins, the induced level of transcripts
achieved in P. glaucus is generally higher than in P. canadensis
(16~. The initial member of this group to be defined functionally,
CYP6B4 from P. glaucus, has the demonstrated capacity to
metabolize linear furanocoumarins (ref. 18 and Table 1~.
Whether P450s paralogous or orthologous to CYP6B4 also
possess these activities was unknown.
To study the evolution of structure and function of P450s
within the context of shifts in host plant utilization and con-
comitant changes in the chemical milieu experienced by the
insect, we compared the CYP6B induction profiles and protein
functionalities in these two species in the context of host plant
furanocoumarin chemistry. For this study, representative P450s
from each of the CYP6B4 and CYP6B17 groups in these species
were expressed in baculovirus expression systems in conjunction
with the insect NADPH P450 reductase needed for full func-
tional activity. Enzymes were tested for their activity and
specificity with respect to host plant furanocoumarins and
related compounds to determine whether reduced probability of
encounter leads to loss-of-function sequence changes consistent
with a birth-and-death process of gene diversification. Also,
ancestral P450 genes were reconstructed by maximum likelihood
methods to chart a theoretical course for this process.
Methods
Construction and Expression of Recombinant Baculovirus. Three P.
glaucus sequences, including the CYP6B4 cDNA and the
CYP6B17 and CYP6B21 genomic DNAs, and two P. canadensis
sequences, including the CYP6B25 and CYP6B26 cDNAs were
expressed by using the baculovirus expression system. For ex-
14594 1 www.pnas.org/c~i/doi/10.1073/pnas.1934643100
pression of the CYP6Bl 7 and CYP6B21 sequences, their 5' UTR
and introns were removed by using a PCR-based strategy before
subcloning them into the pFASTBacl baculovirus expression
vector (Invitrogen). Briefly, this strategy involved amplifying the
5' end 417 bp of the CYP6B17 or CYP6B21 coding sequences
with a forward N1 primer (5'-CCGCTCGAGATCATGTTAA-
CGATATTTAT-3') that contains the start codon and a reverse
C5 primer (5'-CGGCTTAAGTTTTCCTGACGTG-3'), and
inserting the resulting PCR product into pBluescript SK vector.
The remainder of the coding sequence was generated without its
intron by amplifying the C:Ylf~6B17 and CYP6B21 genomic se-
quences with the INTFOR1 primer (5'-CTGGCCAGAGAAA-
ATGCTTAGGAATGCGGTTTGGACAA-3') that spans the
sequences flanking the intron and a reverse C2 primer (5'-
CGGAAGCTTCAATAATTTCGTGGTAAAA-3') and ligat-
ing this PCR product with the 5' coding sequence and the
pBluescript SK vector. The resulting cDNA sequences, which
contain an X7zoI site upstream from the translation start, a
HindIII site downstream from the translation stop and an EaeI
site at the junction between the two fragments, were checked for
amplification errors by sequencing.
All of these CYP6B cDNAs were subcloned into suitable
restriction sites of the pFASTBacl baculovirus expression vector
for construction of recombinant viruses and expressed in the
Bac-to-Bac expression system (Invitrogen). All procedures of
construction and expression of recombinant virus in insect Sf9
cells were performed as described by the manufacturer. P.
polyxenes CYP6B1 cDNA (L. Pan, Z. Wen, J. Baudry, M.R.B.,
and M.A.S., unpublished data) was expressed as a positive
control to monitor P450 expression quality. Sf9 cell cultures were
grown to a density of 0.8 x 106 cells per ml in SF-900 serum-free
medium supplemented with 8-10% FBS, 50 ,ug/ml streptomycin
sulfate, and 50 units/ml penicillin and cotransfected with re-
combinant P450 virus at a multiplicity of infection (MOI) of 2
and recombinant house fly NADPH P450 virus at an MOI of 20
(ref. 19 and L. Pan, Z. Wen, J. Baudry, M.R.B., and M.A.S.,
unpublished data). This MOI ratio of 1:10 was intended to
supplement the limited electron transfer capacities of Sf9 cells.
Li et a/.
OCR for page 83
Hemin was added to 2 ,ug/ml final concentration 24 h after
infection. Insect cells were harvested from ~6 plates for each
P450 and lysed as described by Chen et al. (20), and protein
aliquots were frozen in liquid nitrogen before analysis. Carbon
monoxide (CO) difference spectra were measured as in Omura
and Sato (21) by using an extinction coefficient for the reduced
CO complex of 91 mM-i cam.
P450 Metabolic Activity Assays. In vitro metabolism assays of
furanocoumarins were conducted as described in Li et al. (16)
except that the substrate concentration in each reaction was
lowered to 1 ,ug/ml (corresponding to final concentrations of 4.6
AM xanthotoxin, 4.6 EM bergapten, 5.4 ,uM angelicin, or 5.4 ,uM
psoralen) and a different furanocoumarin was added as an
internal control after reactions were terminated. Rates of O-
deethylation of 7-ethoxycoumarin were determined by measur-
ing the fluorescence of the 7-hydroxycoumarin product (22~. All
metabolic assays were performed with four replicates and re-
peated at least twice by using proteins prepared from indepen-
dent infections. Metabolic activities were compared by analysis
of variance as described (164.
Sequence Alignments, Phylogenetic Analyses, and Reconstruction of
Ancestral Sequences. Amino acid sequences of 22 CYP6B pro-
teins, 12 other CYP6 proteins, and the divergent CYP321A1
protein were aligned to rabbit CYP2C5 (PDB ID 1DT6) and
Bacillus megaterium CYP102 (PDB ID 2HPD), for which crystal
structures are available (23, 24~. Multiple sequence alignments
were performed by using CLUST4W or multialignment modules
within the MOE program (Chemical Computing Group, Mon-
treal) with the Gonnet weight model and structural alignment
enabled. Alignments generated by both methods were compared
and modified according to secondary structures of the CYP2C5
and CYP102 proteins. The finalized alignment was analyzed by
the MEGA program to construct the phylogeny of the CYP6
family. A maximum parsimony phylogeny of CYP6 sequences
was generated by using max-mini branch-and-bound method
and the inferred phylogeny was tested by 500 bootstrap tests. A
maximum likelihood method was used to construct ancestral
CYP6B sequences from the above multiple alignment of protein
sequences on the most parsimonious phylogeny by using PAML
(25) with the implementation of Jones amino acid transforma-
tion model and iteratively estimated fly shape parameter. To
subsequently determine the relationship of ancestral sequences
to their descendant P450s, relative amino acid distances between
the ancestral sequences and their descendant branches were
computed by using the MEGA program based on either complete
protein sequences or active site amino acid residues. Active site
residues were defined, based on our recent furanocoumarin
docking study, as amino acids that are <4.5 ~ from the oxo-heme
moiety or furanocoumarin substrates docked into 3D models of
CYP6B proteins (26~.
Results and Discussion
Catalytic Activity of CYP6B Enzymes. For comparative purposes, five
Papilio CYP6B enzymes, including CYP6B4 (P. glaucus),
CYP6B17 (P. glaucus), CYP6B21 (P. glaucus), CYP6B25 (P.
canadensis), and CYP6B26 (P. canadens~s), were coexpressed
with house fly P450 reductase in Sf9 cells as described in
Methods. The quality and quantity of the expressed P450 proteins
were determined by reduced CO difference analysis (21) using
Sf9 cell lysates prepared from cells cotranfected with recombi-
nant P450 and P450 reductase viruses. In these assays, all of the
P. glaucus enzymes generated CO-difference maxima at 450 nm
(P450 form) with no significant absorbance at 420 nm, indicating
that all of the P450 proteins fold and incorporate heme correctly
into their apoproteins. Of the two P. canadensis proteins,
CYP6B25. an in-group homologue to CYP6B4 (17), generated
Li et a/.
a major CO-difference peak at 450 rim and a minor peak at 420
nm and CYP6B26, an in-group homologue to CYP6B17 and
seven amino acids shorter at its C terminus (16), generated a
CO-difference peak only at 420 nm (P420 form), indicating that
this enzyme is incorrectly folded. Molecular modeling and
site-directed mutagenesis presented in Chen et al. (20) have
indicated that aromatic residues F116, H117, and F484 positions
are required for correct folding of the P. polyxenes CYP6B1
enzyme, and that two of these modulate the range of furano-
coumarins metabolized. This seems not to be the case for P.
glaucus/P. canadensis enzymes that contain a nonaromatic res-
idue (L484) replacing F484 in the P. polyxenes enzyme and
nonetheless generate only functional P450. The low stability of
the CYP6B25 protein, which also contains L484, is apparently
caused by substitutions at other positions because F116, H117,
and L484 are conserved between CYP6B4 and CYP6B25. The
inability of the CYP6B26 protein to fold into stable P450
indicates the importance of C-terminal residues in determining
overall protein folding or configuration of the heme-binding
domain.
To determine the influence of furanocoumarin exposure on
P450 catalytic activity and selectivity, the metabolic activities of
the expressed CYP6B proteins against five linear and angular
furanocoumarins were assayed and compared with the metabolic
activity of the P. polyxenes CYP6B1 protein coexpressed with
P450 reductase in Sf9 cells (Table 1) (27~. The substrates tested
include methoxylated (bergapten, xanthotoxin), trimethylated
(trioxsalen), and unsubstituted (psoralen) linear furanocouma-
rins and an unsubstituted (angelicin) angular furanocoumarin.
These metabolic data indicate that all of the Papilio CYP6B
enzymes are able to turn over furanocoumarin substrates to
some extent, except for the denatured CYP6B26 protein, which
did not metabolize any of the tested substrates (Table 1~.
The relative abilities of the CYP6B proteins to metabolize
linear furanocoumarins were closely associated with the proba-
bility of encountering furanocoumarins in host plants. CYP6B1,
characterized from the specialist P. polyxenes, which encounters
high levels of furanocoumarins in all of its host plants, turns over
linear furanocoumariIls at the highest rate (6980 pmol/min/
nmol P450 for xanthotoxin) among all of CYP6B enzymes tested.
CYP6B4 and CYP6B17, characterized from the generalist P.
glaucus, which occasionally encounters furanocoumarins, turn
over linear furanocoumarin substrates at rates lower than does
CYP6B1 but significantly higher than do CYP6B25 and
CYP61326, characterized from the generalist P. canadensis,
which never encounters furanocoumarins naturally. Further
comparisons of P450 substrate specificities have indicated that
CYP6B1 is more selective than the CYP6B proteins in polyph-
agous P. glaucus/P. canadensis. CYP6B1 exhibits very high
activity toward the methoxylated linear furanocoumarins xan-
thotoxin and bergapten, lower activity toward unsubstituted and
other linear furanocoumarins, and much lower activity toward
the angular furanocoumarin, angelicin (ratio of activity for
xanthotoxin/psoralen/angelicin is 1.0/0.4/0.1) (Table 1) (18,
27~. This finding suggests that linear and angular furanocouma-
rins in P. polyxenes are metabolized by distinct P450s that possess
relatively high substrate specificity. The in-group homologues
CYP6B4 and CYP6B25 display more uniform activities toward
a number of furanocoumarin substrates. Whereas CYP6B4 and
CYP6B25 turn over methoxylated furanocoumarins at higher
rates than other furanocoumarins, they metabolize psoralen and
angelicin at rates higher than those of CYP6B1 (for CYP6B4,
xanthotoxin/psoralen/angelicin is 1.0/0.7/0.6; for CYP6B25, it
is 1.0/0.5/0.3~. Importantly, these ratios demonstrate that these
P450s exhibit the same preference for unsubstituted linear and
angular furanocoumarins in that they turn over psoralen and
angelicin at approximately the same rate. In comparison,
CYP6B17, the paralog of CYP6B4 in P. glaucus, turns over linear
PNAS I November 25, 2003 1 vol. 100 | suppl. 2 | 14595
OCR for page 84
Table 2. Ethoxycoumarin 7-0-deethylation activities of P.
glaucuslP. canadensis CYP6B enzymes
How HI
7-Ed~o:~wcoumann 7-Hydro~vcoumann
P450s CYP6B4 CYP6B17 CYP6B21 CYP6B25 CYP6B26
O-deethvlation*
(pmol/m~n/nmol P450)
143~4 518$4 NDt NDt
*Values listed are mean + SD of at least two independent determinations
each with four replicates.
tNo detectable O-deethylation activity found for CYP6B25 and CYP626.
furanocoumarins at rates lower than CYP6B4 and exhibits no
activity against angular furanocoumarin. Together, these data
indicate that, among the enzymes tested, furanocoumarin me-
tabolism in P. glaucus is mediated primarily by CYP6B4.
Additional assays performed to determine the range of sub-
strates other than furanocoumarins metabolized by CYP6B
enzymes in P. glaucus and P. canadensis demonstrate that at least
one substrate, 7-ethoxycoumarin (EC), representing alkoxylated
coumarins that are widely present in the host plants of swal-
lowtails (12), is metabolized by all three P. glaucus P450s at levels
lower than those defined for furanocoumarin substrates. Inter-
estingly, despite its higher activities against furanocoumarins, the
7-ethoxycoumarin O-deethylase (ECOD) activity of CYP6B4 is
the lowest of all of the P. glaucus P450s tested, with an activity
that is 5.1- and 1.4-fold lower than those of CYP6B21 and
CYP6B17, respectively (Table 2~. In contrast, CYP6B25 and
CYP6B26 from P. canadensis have no detectable ECOD activity.
Recent studies have indicated that the more specialized CYP6B1
from P. polyxenes also has no ECOD activity (Z. Wen, personal
communication).
The catalytic efficiencies and substrate ranges of these CYP6B
proteins map closely onto the range of host plants encountered
by the insect species producing these P450s. In the specialist P.
poly~cenes, which has the highest frequency of encountering
furanocoumarins, the CYP6B enzyme examined is more spe-
cialized and capable of metabolizing furanocoumarins at higher
rates than the CYP6B proteins expressed in other Papilio species.
In the generalist P. glaucus/P. canadensis, the CYP6B proteins
have broader substrate ranges than the CYP6B1 proteins but
metabolize this broader range of substrates at lower rates. The
P. canadensis enzymes have even lower metabolic activities for
all of the substrates tested than those of their P. glaucus
orthologues. These relatively low activities may relate to the
unpredictability of the chemical milieu encountered by P. cana-
densis and P. glaucus compared with the specialist P. polyxenes
and their need to maintain P450 catalytic sites capable of
metabolizing this broad range of substrates. For P. glaucus, which
infrequently encounters these toxins, the low activities probably
result from the lack of selection pressure to maintain furano-
coumarin metabolism. This is particularly evident in P. cana-
densis, which has no natural exposure to furanocoumarins, and
the lower activities of its CYP6B enzymes probably result from
the accumulation of many deleterious mutations that have no
purifying selection driving their elimination. Within P. glaucus,
differences in substrate specificities between the CYP6B4 and
CYP6B17 paralogs are likely to be the consequence of positive
selection for duplicated genes. CYP6B7, CYP6B8, and
CYP6B27, which are derived from Helicoverpa zea/Helicoverpa
armigera, two closely related agricultural pests with extremely
wide host ranges, are inducible by a wide range of chemicals,
including insecticides, plant hormones, and plant allelochemi-
cals, such as xanthotoxin (28-30~. Among these, the CYP6B8
protein has a demonstrated ability to metabolize a range of plant
allelochemicals and insecticides (31), as befits its highly diverse
14596 1 www.pnas.org/cgi/doi/10. 1 073/pnas. 1934643100
5:
| ~ r r~"
CYP1 02
0.5
- CYP2C5
Fig. 1. Reduced representation of the most parsimonious phylogeny of
insect and mammalian P450s. The CYP subfamily was highlighted with gray
background. The non-CYP6B sequences were retrieved from GenBank. The
phylogenetic tree has been drawn proportional to branch lengths calculated
by using maximum likelihood method. The numbers below internal branches
are bootstrap values. The species origin of each CYP6B protein is indicated in
parentheses after each sequence name.
chemical environment. This finding further confirms the asso-
ciation between P450 versatility and host plant range.
Phylogeny of CYP6B Genes and Reconstruction of Aneestral CYP6B
Sequences. Given the close relationship between CYP6B catalytic
efficiency and substrate specificity and the complexity of the
chemical environment experienced by the insect, it is of great
interest to explore how this association was established and how
it evolved into the present-day CYP6B enzymes. For this anal-
ysis, a phylogeny of CYP6B genes was constructed based on the
multiple sequence alignments described in Methods and resulting
in the most parsimonious phylogeny shown in Fig. 1. In this,
CYP6B sequences form a distinct clade with the H. zea
CYP321A1 sequence that is phylogenetically separate from
other CYP6 clades. The close relationship of the CYP321A1
protein to CYP6B proteins is confirmed by similarities in their
function; CYP321A1 is the only non-CYP6B insect P450 known
to be capable of metabolizing furanocoumarins (32~. Within the
CYP6B branch, Papilio and Helicoverpa CYP6B genes form
distinct clades, suggesting no major duplication event before
separation of these species. However, serial duplications oc-
curred in both P. glaucus/P. canadensis and H. zea/H. armigera
branches (Fig. 1), yielding at least 17 closely related CYP6B genes
in the P. glaucus/P. canadensis genomes that do not appear to be
pseudogenes (16, 17~. After the first duplication, two paralogous
groups, designated the CYP6B4-group and the CYP6B17-group
(Fig. 1), formed. Although early studies demonstrated distinct
induction profiles for each of these groups (16), the studies
presented here indicate that members of these groups also
display distinct substrate preferences, indicating functional di-
vergence of these P450s after their initial duplication. The
"redundancy" of CiP6B genes in these polyphagous species is
likely a mechanism for adapting to complex chemical environ-
ments, maintaining one functional gene while another evolves
with different metabolic capabilities. Duplicated copies of P450s
may well provide candidates for subsequent functional diver-
gence to adopt new functions as host plant patterns change.
To characterize the process of functional divergence among
CYP6Bs, three ancestral sequences, the ancestor of the whole
CYP6B subfamily (Ancl), the ancestor of the Papilio branch
(Anc2), and the ancestor of the P. glaucus/P. canadensis branch
~i et a/.
OCR for page 85
O O.S 1 15 2 Z5
1 ~
Papilio lineage > 1
Helicoverpa lineage} Anc1 I
· Active site residues
CYP6B1/6B3} An 2 ~ 0 Complete protein sequence
CYP6B4~B1 7/6B21/6825/6B26 1
CYP6B17/6B26 _
CYP6B4I6~2l/6B2s} Anc3 > 1
Fig. 2. Relative distance of ancestral P450s to CYP6Bs. Grouped bars repre-
sentthe ratio of average amino acid distance between ancestral sequence and
the upper branch to that of the lower branch. The distance ratio was com-
puted either based on active site residues (filled bars) or on the complete
protein sequence (open bars). The ratios indicated as >1 are significantly
different from unity (P < 0.05).
(Anc3), were reconstructed and compared with current CYP6B
proteins (Figs. 2 and 3~. The relative distances (ratios of distance
between ancestral sequences and respective descendant
branches) of all three ancestral sequences are not greater from
unity than those computed based on the complete protein
sequences, suggesting that these genes evolve at approximately
the same rate from their common ancestors (Fig. 2~. Similar
computations were based on functionally more important active
site residues, which directly interact with substrates; the relative
distances of Ancl to Papilio or Helicoverpa sequences, and Anc3
to CYP6B4 group or CYP6B17 group of sequences, respectively,
are statistically different from unity (P < 0.05) (Fig. 2~. Ancl
doe:
Arc:
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sa ace He ~ an an ~ ma ~ an ~ —e ~ ns ~e n n4 ~ ~ —~ ~ _
. ~ ~ A 8 t t S ~ A t V ~ D r L o a ~ a ~ ~ v
A C t t S ~ A F V ~ D ~ L O a ~ a · _ ~
~ ~ t r ~ ~ ~ ~ ._ A_ L _ ~ ~ a L - '
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Fig. 3. A multiple sequence alignment of the amino acid residues in the
active sites of CYP6B and their ancestral P450s. Three ancestral sequences are
shown, the ancestral CYP6B sequence (Anc1), the ancestral Papilio CYP6B
(Anc2) and the ancestral P. glaucus/P. canadensis CYP6B (Anc3). Residue
positions are labeled according to their position in CYP6B4 and CYP6B1. The
residues inside the black boxes represent SRS domains (35). Amino acids
identical to Anc1 were highlighted in yellow; amino acids identical to Anc2 but
not Anc1 are shown in blue; amino acids identical to Anc3, but neither Anc1
nor Anc2, are shown in red. Amino acids that were proposed to be critical to
P450 substrate specificity (Fig. 4) are indicated in bold.
Li et a/.
displayed a significantly closer relationship to Helicoverpa P450s.
The amino acid substitution rate leading to the Papilio lineage is
1.5-fold higher than the rate leading to the Helicoverpa lineage.
It is conceivable that Ancl may have similar functionality to that
of Helicoverpa P450s and very likely catalyzed a broader range
of substrates with lower metabolic efficiency against furanocou-
marins compared with most of the Papilio P450s. Anc2 is
proposed to have existed in a specialist species with constant
exposure to furanocoumarins and to have had relatively higher
catalytic activities against furanocoumarins compared with most
Papilio P450s (17~. This is likely to be the case, because the amino
acid substitution rate of the CYP6B1/CYP6B3 lineage appears
to be 1.3-fold that of the CYP6B4/CYP6B17 lineage, although
this difference is not statistically significant because of higher
sequence variation between CYP6B1/CYP6B3 pair. Anc3 is
more closely related to the CYP6B4 group of P450s than the
CYP6B17 group, as indicated by comparing the postduplication
amino acid substitution rates leading to these lineages. Among
all CYP6B lineages, the CYP6B17 and CYP6B4 branches dis-
played the most dramatic difference (2.1-fold) in the amino acid
replacement rate from the immediate ancestor (Fig. 2~. After the
duplication that led to diversification of the CYP6B4 and
CYP6B17 groups, the CYP6B4 group evolved relatively more
slowly, and thus very likely resembles Anc3 in both sequence and
function; the CYP6B17 group, however, as the duplicate pre-
sumably relieved of purifying selection, evolved faster and
adapted to novel functionality.
Further insight into the evolutionary lineage of these P450
proteins has been gathered by comparing the predicted struc-
tures of these proteins with those predicted for the more efficient
CYP6B1 protein. In this latter protein, an aromatic network that
involves residues Phe-116, His-117, Phe-484, and Phe-371 is
critical for substrate binding affinity to the CYP6B1 active site
(20, 33~. Three-dimensional models of the present day CYP6B4
and the ancestral Ancl and Anc2 proteins that we have con-
Fig. 4. Superposition of the active sites of ancestral CYP6B proteins with
CYP6B1 and CYP6B4. (a) Anc1. (b) Anc2. The amino acid residues of CYP6B1
and CYP6B4 and the ancestral sequences are shown with the residues in the
CYP6B1 sequence or progenitor to it labeled in red and amino acids in the
CYP6B4 sequence or progenitor to it labeled in yellow. Residues involved in
the aromatic interactive network in the CYP6B1 model are displayed as balls
and sticks (33).
PNAS 1 November25, 2003 1 vol. 100 1 suppl. 2 1 14597
OCR for page 86
structed (26) were compared to evaluate the existence and
importance of this aromatic network in defining the stabilities
and substrate specificities of these proteins. The catalytic pocket
of Ancl displayed substantial differences from that of the
CYP6B1 protein (Fig. 4~. Among the four networked residues in
the CYP6B1 model, Phe-116, His-117, and Phe-371 are con-
served in other Papilio CYP6B proteins. In the Ancl model, the
aromatic Phe-116 and His-117 residues are not oriented per-
pendicular to one another in positions typical for aromatic-
aromatic interactions, and the side chain of Phe-371 projects
away from the catalytic pocket in a configuration that may not
allow it to directly interact with other residues in the catalytic
pocket (Fig. 4~. In Ancl, the catalytic pocket is probably larger
in volume and with more flexibility than that defined within the
CYP6B1 protein because a nonaromatic Ile-484 replaces the
larger Phe-484 found in the CYP6B1 protein. In this regard,
Ancl is very similar to H. zea CYP6B8, which is also predicted
to contain a large and flexible catalytic pocket able to accom-
modate a wide range of substrates (31~. The absence and/or
weakening of the ~ T stacking interactions between Phe-484
and furanocoumarin substrates that facilitate binding in the
CYP6B1 catalytic pocket predicts that Ancl probably catalyzed
metabolism of furanocoumarins significantly less efficiently than
the CYP6B1 protein.
In contrast, Anc2 is very likely a CYP6B1-like protein that
maintains the ability to metabolize furanocoumarins. Residues
involved in formation of the aromatic network, Phe-116, His-117,
Phe-371, and Phe-484, are conserved and occupy approximately
identical spatial locations to those in the CYP6B1 protein (Fig.
4b), indicating that the Anc2 enzyme has an aromatic network
that is similar to that in CYP6B1 protein. This might be
presumed to make the catalytic pocket relatively narrow and
rigid, indicating similar structural constraints to that in the
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14598 1 www.pnas.org/cgi/doi/10.1073/pnas.1934643100
CYP6B1 protein, presumably resulting from strong selection by
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In summary, our studies demonstrated for the first time how
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P450s. It is highly likely that insects possessing CYP6B enzymes
originated from a broadly polyphagous species with limited
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furanocoumarin-containing host plants, the ability to metabolize
a broad range of substrates declined as the ability to metabolize
the principal host plant defense compounds, the furanocouma-
rins, reached a level of refinement rarely equaled by other
herbivores. This tradeoff between P450 breadth and specificity
may explain in part the ubiquity of oligophagy that characterizes
lepidopterans today (34~.
We thank Dr. Jerome Baudry for extensive training in modeling pro-
grams and Dr. Liping Pan for providing recombinant baculovirus
containing NADPH P450 reductase. This work has been supported by
National Science Foundation Grant IBN 02 12242 (to M.R.B.) and
National Institutes of Health Grant RO1 GM50007 (to M.A.S.~.
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Li et al.
Representative terms from entire chapter:
catalytic pocket
