From the
Guayule plants accumulate large quantities of rubber within
parenchyma cells of their stembark tissues. This rubber is packed
within discrete organelles called rubber particles composed primarily
of a lipophilic, cis-polyisoprene core, small amounts of
lipids, and several proteins, the most abundant of which is the
M
Rubber is a macromolecular polyisoprenoid found in over 2000
plant species
(1, 2) . It accumulates in discrete,
subcellular organelles called rubber particles that are approximately 1
µm in diameter and are composed of a polyisoprene core
(3, 4) and associated proteins and lipids
(5, 6, 7) . In most rubber-producing species,
such as Hevea brasiliensis, rubber particles are confined to
latex vessels, which are specialized plant cells devoted to latex and
secondary product synthesis. However, in the North American desert
shrub, guayule ( Parthenium argentatum), rubber particles
accumulate within ordinary stembark parenchyma cells
(4) .
Consequently, its rubber is produced in otherwise normal cells,
presenting a unique opportunity for study. Hevea, the
commercial source of virtually all natural rubber, produces particles
that contain dozens of proteins. Some of these proteins can trigger
severe allergies in people who come in contact with products
manufactured from Hevea latex. In contrast, guayule particles
possess few proteins
(8, 9) , and none are known to
cause allergies, making them a potential alternate source of latex
products for medical applications
(10, 11
A major
effort is currently underway to identify the function of
rubber-associated proteins in plants. From a biochemical perspective,
this is less complex for guayule because its particles contain
relatively few proteins. The most abundant is the M
In this report, we now show that
RPP is a member of the CYP74 family of cytochromes P450 and is likely
not a prenyltransferase. This is based on the deduced amino acid
sequence of RPP cDNAs isolated from a guayule stembark cDNA library.
RPP shows significant sequence identity to the P450 known as allene
oxide synthase (AOS)
(13) . Additional biochemical analysis of
partially purified RPP solubilized from washed rubber particles reveals
a difference spectrum typical of P450s and, like AOS, an ability to
rapidly metabolize hydroperoxylinoleic acid into
A large scale reaction was prepared using 20 µl of
CHAPS-solubilized RPP (7.2 µg of protein) and 2 mg of
13 S-HPOD in 6 ml of 50 m
M potassium phosphate buffer
(pH 7.0) incubated at 22 °C for 60 min. The products of the AOS
reaction were extracted with 1.5 volumes of a 2:1 (v/v) mixture of
chloroform:methanol. The chloroform-extracted products were separated
as the free fatty acids on precoated, silica gel TLC plates (T-6270,
Sigma) according to the method of Gardner
(28) using
isooctane:ether:acetic acid (50:50:1, v/v/v) with detection by
2,4-dinitrophenylhydrazine spray
(29) . For isolation, the
products were methyl-esterified by a brief exposure to diazomethane in
diethyl ether:CH
NH
The full-length RPP cDNA contained 1692
nucleotides, including 23 nucleotides of 5`- and 250 nucleotides of
3`-untranslated sequence. It encoded a protein of 473 amino acids, with
an NH
A search of the
genetic sequence data base identified two small regions in RPP that
shared significant homology with two small regions of several
cytochrome P450s
(32, 33) . Those regions aligned with
two highly conserved B and C domains
(32) , which are common to
all P450s (Fig. 2). Additionally, RPP contained a conserved PPGP
tetrapeptide near the amino terminus (Fig. 1) that is also common
in P450s and is known to participate in P450 stability and catalysis
(34) . However, RPP did not contain the highly conserved A and D
domains that are essential components of most P450s. Careful comparison
of the RPP sequence with that of CYP73, a plant P450 known as cinnamic
acid hydrolase (CA4H)
(35) , suggested that the CAG of RPP could
act as a putative heme-binding site necessary for P450 action. However,
the amino acid sequence surrounding this CAG (Fig. 1) was
inconsistent with the known P450 consensus decapeptide of
F-(SGNA)- X-(GD)- X-(RHPT)- X-C-(LIVMFAP)-(GAD)
(32) . This, together with the absence of the A domain,
suggested that RPP might not act as a true P450. Thus, studies were
performed to verify whether RPP possessed the spectral characteristics
of P450s. RPP solubilized from washed rubber particles did, in fact,
yield a difference spectra for the CO-bound, dithionite-reduced form
that clearly indicated that it was a P450 with an absorbance maximum at
451 nm (Fig. 3).
AOS, also previously known as hydroperoxide isomerase
(36, 37) , hydroperoxide cyclase
(38) , fatty
acid hydroperoxide dehydrase
(39) , and hydroperoxide
dehydratase (EC 4.2.1.92), is classified as a hydroperoxide-dependent
P450. It metabolizes lipid hydroperoxides into their corresponding
allene epoxides by intramolecular oxygen transfer
(39) . Enzyme
analysis was performed using RPP solubilized from washed rubber
particles. SDS-PAGE gels indicated that the preparations were greatly
enriched for RPP (Fig. 5). The AOS activity of the CHAPS-solubilized
preparation (Fig. 5, lane 3) had an estimated
k
GC-MS of the
The
GC-MS of the
Another product,
identified as 12-oxo-13-methoxy-9-octadecenoic acid (data not shown),
was isolated by TLC ( R
As with AOS
(13, 14, 41, 42) ,
RPP does not require molecular oxygen or a NADPH-dependent cytochrome
P450 reductase for its activity. RPP also lacks the highly conserved
threonine that is located in the I-helix region of the A domain of
typical P450s. This threonine is thought to participate in substrate
binding and oxygen stabilization and to donate a proton to the
FeO22+ complex. In P450
Nearly all
of the plant cytochrome P450s so far described are microsomal and occur
in relatively low abundance in plant tissues
(45, 46) .
In contrast, RPP is not associated with microsomes and is quite
abundant, comprising the majority of protein found in rubber particles.
Guayule can accumulate extraordinarily large numbers of rubber
particles
(8, 9, 15) , and it suggests that this
particular P450 may have important functions in this species.
AOS is
a member of the lipoxygenase pathway
(40) , a metabolic route
that synthesizes important mediators of plant metabolism. In the
standard series of reactions, linoleic or linolenic acid is oxygenated
by one or more lipoxygenases that produce lipid hydroperoxides. AOS
then converts these potentially toxic compounds into allene oxide,
which is an unstable lipid epoxide
(13, 14, 39, 40, 41) that is
converted into
The RPP in guayule particles has high
AOS activity. However, unlike flaxseed AOS
(14) , RPP is
produced in relatively large quantities. We do not know why high
concentrations of such an active enzyme are present in rubber
particles, but it raises a number of interesting questions with regard
to the origin and function of rubber in plants. It has long been
theorized
(1, 2) that rubber particles provide a
physical barrier to insect feeding. However, they have never been
implicated as a source of enzyme that might produce metabolites that
could deter insect feeding or play a role in plant stress responses. If
RPP is involved in such reactions, it would require access to the
appropriate oxygenated lipids. In fact, this is possible as it has been
reported that Hevea rubber particles contain lipids
(5, 6, 7) that could serve as intermediates for
such reactions. Preliminary evidence has also revealed these lipids
appear in guayule particles and that high lipoxygenase activity occurs
in guayule tissues.
Another possibility is
that RPP is involved in rubber biosynthesis by some unknown mechanism.
This is supported by recent experiments showing that antibodies that
recognize RPP also suppress rubber transferase activity in vitro (49, 50, 51) . These antibodies, originally
prepared against a rubber-associated protein designated LPR (for the
large protein from rubber) from Ficus elastica, also show
strong antigenicity to RPP and a similar M
A
prior study suggested that RPP was rubber transferase
(12) . If
true, this would predict a direct involvement for RPP as a
prenyltransferase. However, the deduced amino acid sequence of RPP does
not share homology with any of the known prenyltransferases. These all
contain two characteristic DD XXD motifs
(52) that are
not present in RPP. It is possible that RPP is involved in other
ancillary reactions, perhaps as a multiprotein complex, which protects
or permits the unusual cis-1,4-polyisoprene polymerizations to
occur. This could potentially explain why antibodies directed against
RPP also suppress prenyltransferase activity in vitro.
Taken together, our results demonstrate that RPP is a new cytochrome
P450 that has been classified as CYP74A2
(33) with sequence and
biochemical activities similar to AOS. Like AOS, RPP is a
hydroperoxide-dependent P450 with remarkably high catalytic turnover
that does not require molecular oxygen or a NADPH-cytochrome P450
reductase for its activity.
The pmol
yield for each cycle is indicated. Inconclusive assignments are
indicated in parentheses.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) X78166.
We thank M. Long for technical assistance, E. Herman,
M. Kuntz, and B. Vick for valuable technical advice, D. Weisleder for
NMR data, and C. Bailey and N. Goodman for support. We also thank D. R.
Nelson of the Cytochrome P450 Nomenclature Committee for help in
assigning RPP to a P450 gene family.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
53,000 rubber particle protein (RPP). We have
cloned and sequenced a full-length cDNA for RPP and show that it has
65% amino acid identity and 85% similarity to a cytochrome P450 known
as allene oxide synthase (AOS), recently identified from flaxseed. RPP
contains the same unusual heme-binding region and possesses a similar
defective I-helix region as AOS, suggesting an equivalent biochemical
function. Spectral analysis of solubilized RPP verifies it as a P450,
and enzymatic assays reveal that it also metabolizes
13( S)-hydroperoxy-(9 Z,11 E)-octadecadienoic
acid into the expected ketol fatty acids at rates comparable with
flaxseed AOS. RPP is unusual in that it lacks the amino-terminal
membrane anchor and the established organelle targeting sequences found
on other conventional P450s. Together, these factors place RPP in the
CYP74 family of P450s and establish it as the first P450 localized in
rubber particles and the first eukaryotic P450 to be identified outside
endoplasmic reticulum, mitochondria, or plastids.
53,000 rubber particle protein (RPP),
(
)
which has been purified
(8) . It comprises
approximately 50% of the protein in guayule particles and has been
implicated as a rubber transferase
(12) , the enzyme that
catalyzes the polymerization of thousands of isoprenes into molecules
of rubber
(1, 2) .
- and
-ketol
fatty acids
(14) . RPP and AOS share a number of significant
features that distinguish them from conventional P450s. However, RPP
also has several unique characteristics that differentiate it from
flaxseed AOS.
Isolation of Rubber Particles
Guayule,
P. argentatum Gray, line 11591, was used for all experiments.
Tissues were collected from field-grown shrubs at the U. S. Department
of Agriculture (Water Conservation Laboratory, Phoenix, AZ). Rubber
particles were purified by flotation from homogenized stembark tissues
(15) .
CNBr Cleavage and Amino Acid Sequencing of
RPP
Particles were subjected to preparative SDS-PAGE
(8) . RPP migrated as a single M53,000
band that was purified from the gel by electroelution (model 422
Electroeluter, Bio-Rad) according to the manufacturer's
instructions. Purified RPP was subjected to CNBr cleavage by dissolving
approximately 1 nmol of lyophilized protein in 150 µl of 70% formic
acid followed by 100 µl of 70 µg of CNBr ml
in 70% formic acid. The mixture was incubated in the dark at room
temperature for 24 h, and the peptide fragments were separated on a 16%
acrylamide gel
(16) and blotted onto polyvinylidene difluoride
(Millipore) membranes
(17) . Stained bands were subjected to
amino-terminal sequencing, performed at the Protein Structure Lab (U.
C. Davis), using an ABI 470A gas phase sequenator. Four peptide
fragments yielded the following amino acid sequences: 1,
PLTKSVVYESLRIEPPV; 2, EQAEKLGVPKDEAVHNILFAVCFNTFGGVK; 3,
LFGYQPFATKDPKVFDRPEEFVPDRFVGDGEALLKY; 4, LKNSSNRVIPQFETTYTELFEGLEA.
Construction of the
Total RNA was isolated from guayule stembark
tissues using the procedure of Logemann et al. (18) .
Tissues were harvested between November and March when rubber synthesis
is highest. Poly(A)ZAP Guayule Stembark cDNA
Library
RNA was used to construct the
ZAP stembark cDNA library according to Short et al. (19) following the manufacturer's instructions
(Stratagene). The library yielded 3
10
recombinants
before amplification. Approximately 1.5
10
plaque-forming units from the original cDNA library were
screened.
Polymerase Chain Reactions
Polyadenylated
RNAs purified from stembark were used for first-strand cDNA synthesis
(20) . Reactions were carried out in a 100-µl total volume
containing 5 µg of poly(A)RNA, 1 m
M dNTPs, 0.1 µg of oligo(dT)
(Amersham
Corp.), 1 unit of RNase block II, and 100 units of M-MuLV reverse
transcriptase (Stratagene). Polymerase chain reactions were carried out
in a 100-µl total volume using 1-2 units of Replinase (DuPont
NEN) or AmpliTaq (Perkin-Elmer Corp.) with the following components: 1%
of first-strand cDNA product, 50 pmol of each primer, 250 µ
M dNTPs in PCR buffer using 30 cycles (1 min at 94 °C, 1 min at
45 °C, and 1 min at 72 °C). PCR reactions used degenerate
primers specific for RPP peptide fragment 3 (sense strand P5,
5`-TTYGGNTAYCARCYNTTYGC-3`; and antisense strand P6,
5`-GCYTCNCCRTCNCCNACRAA-3`). Coding redundancies are: N = C, T,
A, or G; H = A, C, or T; S = G or C; R = A or G; B
= C, G, or T; K = G or T; Y = C or T; V =
A, C, or G; W = A or T; M = A or C; D = A, G or T.
The 92-bp product was sequenced according to Kretz et al. (21) to verify that it matched the sequence for peptide fragment
3. A second pair of primers (sense strand P1, 5`-ATHCYNCARTTYGARAC-3`;
and antisense strand P9, 5`-TTNACNCCNCCRAANGTRTTRAA-3`) produced a
434-bp product that also matched the upstream portion of RPP. These 92-
and 434-bp RPP probes were used to screen the cDNA library.
cDNA Library Screening
Plaque lifts were
prepared using Colony/Plaque Screen (DuPont NEN) and probed with a
[-
P]dATP-labeled PCR fragment
(22) .
Hybridizations were carried out at 65 °C for 16 h in 5
SSC
(1
SSC is 0.15
M NaCl, 0.015
M sodium
citrate), 50 m
M sodium phosphate (pH 7.4), 5
Denhardt's solution (1
Denhardt's solution is 0.02%
Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.5%
SDS, 10% dextran sulfate. Filters were washed four times as follows: 1)
10 min, 2
SSC at room temperature; 2) 10 min, 2
SSC, 1%
SDS at room temperature; 3) 30 min, 2
SSC, 1% SDS at 65 °C,
and 4) 10 min, 0.1
SSC, 0.1% SDS at room temperature. The
filters were exposed to Kodak x-ray films with intensifying screens at
-80 °C. Plaques giving the strongest hybridization signals
were isolated and rescreened under the same conditions. Phagemids were
obtained from purified positive plaques through in vivo excision as described in the manufacturer's protocol
(Stratagene). Over 30 RPP cDNAs were isolated to yield four full-length
clones, one of which is the clone described herein. All sequencing was
by the dideoxy technique
(23) using Sequenase (U.S. Biochemical
Corp.). Sequence comparisons to the genetic data base were made using
the BLAST algorithm
(24) .
Solubilization of RPP from Rubber Particles and P450
Spectral Analysis
Rubber particles isolated from stembark
homogenates were purified by washing at least three times using the
flotation and centrifugation procedure
(15) , except that 50
m
M potassium phosphate (pH 7.0) was used as the extraction and
wash buffer in place of Tris-HCl. RPP was solubilized by sonicating
3-washed particles in 0.5% CHAPS for 1 min. This was centrifuged
at 14,000
g for 5 min, and the solution beneath the
rubber layer was removed and passed through a Millex-SLGV 0.22-µm
filter (Millipore). This eliminated all remaining traces of rubber and
yielded a clear solution containing the solubilized RPP. Each step of
the protein isolation was monitored by SDS-PAGE using 0.75-mm slab gels
of 10% (w/v) polyacrylamide stained with Coomassie Blue. Scanning
densitometry of the Coomassie-stained gels indicated that the RPP band
comprised about 50% of the total protein in rubber particles. The
clarified RPP solution was used for P450 spectral analysis and enzyme
assays. The concentration of total protein in this clarified
preparation was 363 ng µl
based on a comparison
against a bovine serum albumin standard. Additional two-dimensional gel
electrophoresis revealed that the RPP band contained a single
polypeptide and not a mixture of polypeptides. P450 difference spectra
were obtained by diluting the CHAPS-solubilized RPP preparation in 50
m
M potassium phosphate (pH 7.0) and distributing it to 3-ml
reference and sample cuvettes. Both cuvettes were reduced with
Na-dithionite, and the sample cuvette was bubbled with CO to produce a
difference spectrum with a characteristic peak at 451 nm. The fully
developed chromophore appeared after 5 min. The quantity of P450 in the
solubilized RPP preparation, determined from the differential
absorption of the CO-bound reduced form of P450, using the value of
A
- A
= 91 m
M cm
(26, 27) , was 0.16 pmol
µl
or 36.7 ng µl
. Using
this value, the content of P450 per total protein was approximately
10%. Comparing this to the densitometry values, we estimated that the
range of P450/mg of protein in the particles was between 10-50%.
AOS Activity of RPP Solubilized from Rubber
Particles
To test whether RPP was capable of metabolizing
lipid hydroperoxides, the same filtered, CHAPS-solubilized preparations
were used. AOS activity was measured spectrophotometrically
(14) using purified
13( S)-hydroperoxy-(9 Z,11 E)-octadecadienoic
acid (13 S-HPOD) that was prepared according to Ref. 25. The
reaction was initiated by adding 3 µl of CHAPS-solubilized RPP to 3
ml of 50 m
M potassium phosphate buffer (pH 7.0) containing
13 S-HPOD at a final concentration of 53.8 µ
M (1.4
absorbance units at 234 nm). The quantity of P450 in this solubilized
RPP preparation was 0.48 pmol based on P450 difference spectra
(26, 27) . AOS activity was measured by a series of UV
spectra taken at 10-s intervals, which showed the rapid degradation of
substrate due to the loss of its conjugated diene. One-half of the
substrate disappeared after 45 s, resulting in an estimated
kof 3700 s
for this enzyme.
OH (9:1, v/v) and separated by TLC (20
20
0.025 cm, silica 60 F-254 plates, Merck) using
development by hexane:ethyl ether (3:2, v/v). Detection was with a
non-destructive spray, 0.1% 8-anilino-1-naphthalenesulfonic acid (Na
salt) followed by long UV viewing. Fluorescent bands were scraped and
extracted with ethyl acetate. The recovered products were examined by
nuclear magnetic resonance (NMR),
H-NMR and/or
C-NMR, using a Bruker model ARX-400 spectrometer
(Karlsruhe, Germany) with the samples dissolved in CDCl
(CDCl
served as internal reference). Gas
chromatography-mass spectrometry (GC-MS) was completed by a Hewlett
Packard model 5890 (capillary column, Hewlett Packard HP-5MS
cross-linked 5% phenylmethyl silicone, 0.25 mm
30 m, film
thickness 0.25 mm) interfaced with a model 5971 mass selective detector
operating at 70 eV. The temperature program was from 160 to 260 °C
at 5 °C min
with a hold at 260 °C for 10
min. The helium flow rate was 0.67 ml min
. All
products were examined by GC-MS as their methyl ester/trimethylsilyloxy
(OTMS) derivatives. OTMS derivatives of hydroxy groups were synthesized
with trimethylchlorosilane:hexamethyl-disilazane:pyridine (3:2:2,
v/v/v). Other derivatives for GC-MS were produced by either NaBH
reduction of the ketone group or reduction of the double bond by
H
with a 5% palladium on CaCO
catalyst followed
by treatment with OTMS reagent.
-terminal Edman sequencing was performed on
four CNBr fragments of gel-purified RPP (Table I). RPP cDNA was cloned
using a strategy based on these amino acid sequences. Degenerate
oligonucleotide primers were used to produce RPP-specific DNA fragments
by PCR. These were used as hybridization probes to screen our cDNA
library and provided over 30 clones, four of which contained putative
full-length sequences.
-terminal methionine, a COOH-terminal isoleucine, and
two internal cysteines (Fig. 1). The deduced M
53,438 protein contained the four predicted CNBr peptide
fragments, and its deduced isoelectric point (pI 6.15) matched the
experimental value (pI 6.2) of purified RPP
(8) . A comparison
of the deduced and measured amino acid composition
(8) was also
in agreement. Primer extension analysis revealed that RPP mRNA extended
about 35 nucleotides upstream from the 5`-end of the RPP cDNA (data not
shown), indicating that the cDNA was a full-length clone for RPP. The
sequence flanking the first AUG initiation codon (5`-AAAACAUGG-3`)
represented high homology to the consensus AUG start site
(5`-TAAACAAUGG-3` and 5`-AAAA(A/C)AUGG-3`) for translation initiation
in plants
(30, 31) , providing additional evidence that
this cDNA contained the entire RPP-coding sequence.
Figure 1:
Nucleotide and amino acid
sequence of the guayule stembark cDNA for RPP. Shaded areas indicate the CNBr fragments of RPP purified from
rubber particles. The position of the degenerate oligonucleotide
primers P5, P6 and P1, P9 are indicated. These were used to prepare the
RPP-specific probes by PCR, which were used to screen the cDNA library.
The A, B, C, and D domains of conventional cytochrome P450s are
enclosed in boxes. The PPGP tetrapeptide near the amino
terminus is indicated ( asterisks). The position of
I, which replaces the conserved threonine of the I-helix
in domain A, is designated with an arrow. The modified
decapeptide including the CAG ( underlined) of the heme-binding
site in domain D is also denoted (+).
Subsequent to this, we discovered that RPP had
significant homology with the amino acid sequence for AOS from
flaxseed, whose cDNA was recently described
(13) . RPP showed
over 65% identity and 85% similarity to AOS, which is now known to be
an atypical P450, classified as CYP74
(33) . AOS, noted for its
unusual heme-binding site
(13) , possesses a 15-amino acid
peptide sequence that is nearly identical to the putative heme-binding
CAG region of RPP (Fig. 4). This sequence, beginning at RPP position
419, shows over 86% identity with the corresponding flaxseed AOS
sequence (Fig. 4) and hints that RPP and AOS might catalyze similar
reactions.
of 3700 s
, as determined by
scanning UV spectroscopy. This is within the same order of magnitude as
flaxseed AOS, which has a k
of approximately
1000 s
(14) . Analysis of the reaction
products by TLC, NMR, and GC-MS also confirmed that RPP had AOS
activity. These products consisted of the same
- and
-ketol
fatty acids that are observed with flaxseed
(14, 36, 37, 38) and corn AOS
(28, 39) . The
- and
-ketols result from the
rapid degradation of allene oxide produced by the AOS reaction.
Figure 5:
SDS-PAGE for each step leading to
solubilized RPP as detected by Coomassie Blue staining of 10%
polyacrylamide gels. Lane 1, initial guayule stembark
homogenate; lane 2, 3-washed rubber particles
collected by flotation and centrifugation; lane 3,
solubilized RPP prepared by sonicating washed rubber particles for 1
min in 0.5% CHAPS and filtered to remove all remaining rubber
particles. M, molecular size markers (sizes shown at left in
kilodaltons). Arrow indicates RPP
band.
The
-ketol, as its methyl ester, was isolated by TLC
( R
= 0.34), affording a 56% yield
based on the amount of utilized 13 S-HPOD (1.96 mg).
H-NMR of the
-ketol (methyl ester) furnished the
following data in chemical shifts in ppm and number of protons,
multiplicity, coupling constants and carbon assignments: 5.62 (1H,
dt, J
= 10.8 Hz;
J
= 7.3 Hz, C-10 or -9), 5.51 (1H,
dt, C-9 or -10), 4.23 (1H, m, C-13), 3.67
(
H, s, OCH
), 3.40 (1H, d,
J = 5.0 Hz, OH at C-13), 3.22 and 3.24 (1H each,
d, J
= 7.3 Hz, C-11a, b), 2.29
(2H, t, J = 7.5 Hz, C-2), 2.01 (2H,
m, C-8), 1.48 and 1.81 (1H each, m, C-14a, b), 1.60
(2H, m, C-3), 1.30 (14H, m, C-4 to -7 and C-15 to
-17), 0.88 (
H, t, C-18). The assignments were
confirmed by two-dimensional proton correlative spectroscopy. A
significant feature of the
H-NMR spectrum was the coupling
J
= 10.8 Hz establishing the
( Z) configuration of the double bond. An unusual splitting of
the C-13 OH proton was due to hydrogen bonding to the vicinal ketone,
which inhibited the normal hydrogen exchange and lack of coupling of
hydroxyl hydrogens. The
C-NMR of the
-ketol (methyl
ester) furnished an absorbance due to the C-12 ketone at 210.5 ppm
distinct from the other downfield absorbances of C-1 at 174.0 ppm and
C-9 and -10 at 119.7 and 134.3 ppm, respectively.
-ketol, as its methyl ester OTMS derivative, gave four peaks with
similar mass spectra indicating double bond isomerization from reagent
treatment or GC. The largest of these gave the following mass spectrum:
[ m/z (% relative intensity, ion structure)] 383 (3,
[M - CH
]
), 367 (0.4,
[M - CH
O]
), 299 (8,
[M - HOTMS]
), 270 (17, rearrangement
[CH
- CH =
CH(CH
)
COOCH
+
TMS]
), 173 (100,
[CHOTMS(CH
)
CH
]
),
73 (48, TMS
). On the other hand, hydrogenation of the
-ketol followed by OTMS derivativization gave one GC peak, as
expected, with the following mass spectrum: 400 (0.1,
M
), 385 (2, [M -
CH
]
), 272 (13, rearrangement
[(CH
)
COOCH
+
TMS]
), 173 (100,
[CHOTMS(CH
)
CH
]
),
103
(14) ; 73 (50, TMS
). NaBH
reduction of the
-ketol or its hydrogenation product
afforded two GC peaks of equal size due to erythro- and
threo-isomers of the vicinal diol. Both GC peaks gave
identical mass spectra consistent with that expected. The
NaBH
-reduced derivative furnished the most informative mass
spectrum: 457 (1, [M -
CH
]
), 441 (2, [M -
CH
O]
), 382 (1, [M -
HOTMS]
), 299 (34, [M -
CHOTMS(CH
)
CH
]
),
275 (38,
[CHOTMSCHOTMS(CH
)
CH
]
),
270 (18, rearrangement [CH
- CH =
CH(CH
)
COOCH
+
TMS]
), 185 (9, [275 -
HOTMS]
), 173 (67,
[CHOTMS(CH
)
CH
]
),
147
(24) ; 103
(15) ; 73 (100, TMS
). The
NaBH
-reduced hydrogenated derivative gave three principal
ions: 301 (76, [M -
CHOTMS(CH
)
CH
]
),
173 (100,
[CHOTMS(CH
)
CH
]
),
and 73 (95, TMS
). Together, the above data firmly
established the product as
-ketol.
-ketol (methyl
ester) was recovered from TLC as a UV-absorbing band
( R
= 0.11) in 15% yield based on
starting substrate. Its
H-NMR furnished the following data:
6.77 (1H, dd, J
= 15.9 Hz,
J
= 5.0 Hz, C-10), 6.29 (1H, d,
C-11), 4.30 (1H, m, C-9), 3.66 (
H, s,
OCH
), 2.54 (2H, t, J
= 7.5 Hz, C-13), 2.29 (2H, t, J
= 7.5 Hz, C-2), 1.58 (m, C-3, -8, -14), 1.30
(m, C-4 to -7 and C-15 to -17), 0.87 (
H, t, C-18).
The coupling constant, J
= 15.9 Hz,
established the ( E) configuration of the unsaturation. The
multiplicity of the C-11 olefin indicated that it was vicinal to a
ketone, whereas the olefin multiplicity at C-10 indicated it was
vicinal to a secondary alcohol.
-ketol, as its
methyl ester OTMS derivative, gave the following mass spectrum
[ m/z (% relative intensity, ion structure)]: 398 (2,
M
), 383 (3, [M -
CH
]
); 285
(22) , 270
(18) , 259 (10,
[CHOTMS(CH
)
COOCH
]
),
241 (100, [M -
(CH
)
COOCH
]
),
129
(32) , 73 (98, TMS
). The mass spectrum of
the NaBH
-reduced and hydrogenated
-ketol (OTMS, methyl
ester) established two hydroxylated substituents at C-12 and C-9 as
follows: 385 (0.3, [M - HOTMS]
), 360
(4, rearrangement
[(CH
)
CHOTMS(CH
)
COOCH
+ TMS]
), 317 (7, [M -
(CH
)
COOCH
]
),
299 (72, [M - CH
(CH
)
- HOTMS]
), 227 (93,
[317-HOTMS]
), 187 (85,
[CH
(CH
)
CHOTMS]
),
129
(49) , 73 (100, TMS
).
= 0.58) in
10% yield. This compound could have been formed either by diazomethane
treatment of the
-ketol or by solvolysis of newly formed allene
oxide by addition of CHCl
:CH
OH for extraction.
(43) , this threonine
is found at position 252 and at position 310 of CA4H (Fig. 4). In
both RPP and flax AOS, it is replaced with isoleucine at position 283
in RPP and at position 346 in AOS and suggests an altered I-helix
function for both enzymes. A comparison of hydropathy plots for RPP and
AOS reveals greater similarity between these two proteins than either
compared with CA4H, a more conventional plant P450 (Fig. 6).
Figure 4:
Comparison of the amino acid sequence of
RPP with flaxseed AOS and CAH4 (CYP73). RPP shows 65% identity and 80%
similarity with AOS but much less overall homology to CAH4, a typical
microsomal plant P450 (35).
RPP has
a number of differences with flaxseed AOS. Most notable is that RPP is
60 amino acids shorter at its amino terminus than AOS. In AOS, this
segment contains the putative transit sequence that is thought to
target it to plastids or mitochondria
(13) . Biochemical
evidence for spinach AOS corroborates its localization in chloroplast
membranes
(44) . In contrast, RPP lacks this 60-amino acid
sequence, and, coincidentally, the enzyme is localized in rubber
particles. Biochemical assays indicate that AOS activity is absent in
other non-rubber particle-containing fractions of guayule stembark
extracts (data not shown). It is not known if the absence of the
60-amino acid amino terminus in RPP is responsible for its localization
in rubber particles. Until now, rubber particles have not been
associated with any P450 activity. It remains to be seen whether rubber
particles of other species contain P450 or AOS activity.
- and
-ketols and a cyclopentenyl metabolite.
The cyclopentenyl product is a metabolic precursor of jasmonic acid, a
prostaglandin-like plant growth regulator. Jasmonic acid and its
derivatives are known to act as chemical messengers during plant stress
and insect defense
(47, 48) . It is not known if rubber
plays a role in this process.
(
)
protein
from Hevea. LPR is a high M
glycoprotein
that, like RPP, is the most abundant protein of Ficus particles. It has been suggested that RPP and LPR form large
multiprotein complexes within their respective rubber particles and, in
this way, participate in rubber biosynthesis
(51) . Evidently,
rubber particles from these other species may also contain P450s.
Table: N-terminal sequence analysis of the CNBr
fragments from the guayule rubber particle protein
-ketol, 12-oxo-13-hydroxy-9( Z)-octadecenoic acid;
-ketol, 12-oxo-9-hydroxy-10( E)-octadecenoic acid; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid; GC-MS, gas chromatography-mass spectrometry; OTMS,
trimethylsilyloxy derivative; AOS, allene oxide synthase; bp, base
pair(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.