©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Major Protein of Guayule Rubber Particles Is a Cytochrome P450
CHARACTERIZATION BASED ON cDNA CLONING AND SPECTROSCOPIC ANALYSIS OF THE SOLUBILIZED ENZYME AND ITS REACTION PRODUCTS (*)

Zhiqiang Pan (1), Francis Durst (2), Daniele Werck-Reichhart (2), Harold W. Gardner (3), Bilal Camara (4), Katrina Cornish (5), Ralph A. Backhaus (1)(§)

From the (1) Department of Botany, Arizona State University, Tempe, Arizona 85287-1601, (2) CNRS, Institut de Biologie Moleculaire des Plantes, Departement d'Enzymologie Cellulaire et Moleculaire, Université Louis Pasteur, Institut de Botanique, 28 rue Goethe, F-67083, Strasbourg, France, the (3) United States Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61064, (4) CNRS, Institut de Biologie Moleculaire des Plantes, 12 rue du General Zimmer, F-67084, Strasbourg, France, and the (5) United States Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Process Biotechnology, Albany, California 94710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 M53,000 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.


INTRODUCTION

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 M53,000 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) .

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 - 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.


EXPERIMENTAL PROCEDURES

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 mlin 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 ZAP Guayule Stembark cDNA Library

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)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 10recombinants before amplification. Approximately 1.5 10plaque-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 µlbased 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 µlor 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 sfor this enzyme.

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:CHOH (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(CDClserved 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 minwith 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 NaBHreduction of the ketone group or reduction of the double bond by Hwith a 5% palladium on CaCOcatalyst followed by treatment with OTMS reagent.


RESULTS

NH-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.

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-terminal methionine, a COOH-terminal isoleucine, and two internal cysteines (Fig. 1). The deduced M53,438 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.

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).


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.

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 kof 3700 s, as determined by scanning UV spectroscopy. This is within the same order of magnitude as flaxseed AOS, which has a kof 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.

GC-MS of the -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 - CHO]), 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). NaBHreduction 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 - CHO]), 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.

The -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.

GC-MS of the -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).

Another product, identified as 12-oxo-13-methoxy-9-octadecenoic acid (data not shown), was isolated by TLC ( R= 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:CHOH for extraction.


DISCUSSION

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(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.

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 - 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.

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 Mprotein from Hevea. LPR is a high Mglycoprotein 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.

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.

  
Table: N-terminal sequence analysis of the CNBr fragments from the guayule rubber particle protein

The pmol yield for each cycle is indicated. Inconclusive assignments are indicated in parentheses.



FOOTNOTES

*
This research was supported by National Science Foundation Grant MCB-92-20417 and United States Department of Agriculture Grant CSRS-90-38200-5568 (to R. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X78166.

§
To whom correspondence should be addressed. Tel.: 602-965-5564; Fax: 602-965-6899; E-mail: atrab@acvax.inre.asu.edu.

The abbreviations used are: RPP, rubber particle protein; 13 S-HPOD, 13( S)-hydroperoxy-(9 Z,11 E)-octadecadienoic acid; -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).

Z. Pan and R. A. Backhaus, unpublished results.


ACKNOWLEDGEMENTS

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.


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