©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Bacterial Expression of a Sesquiterpene Cyclase from Hyoscyamus muticus and Its Molecular Comparison to Related Terpene Cyclases (*)

(Received for publication, January 10, 1995)

Kyoungwhan Back Joseph Chappell (§)

From the Plant Physiology/Biochemistry/Molecular Biology Program, Agronomy Department, University of Kentucky, Lexington, Kentucky 40546-0091

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Genomic and cDNA clones for vetispiradiene synthase, a sesquiterpene cyclase found in Hyoscyamus muticus, were isolated using a combination of reverse transcription-polymerase chain reactions and conventional cloning procedures. RNA blot hybridization demonstrated an induction of mRNA consistent with the induction of cyclase enzyme activity in elicitor-treated cells, DNA blot hybridization indicated a gene family of 6 to 8 members, and bacterial expression of 3 cDNA clones indicated that each coded for a vetispiradiene synthase enzyme activity catalyzing the synthesis of a single reaction product. Intron-exon organization of the vetispiradiene synthase gene was identical with that previously described for 5-epi-aristolochene synthase (tobacco sesquiterpene cyclase) and casbene synthase (castor bean diterpene cyclase), and the vetispiradiene synthase amino acid sequence was 77% identical with and 81% similar to the tobacco sesquiterpene cyclase. Regions of the vetispiradiene synthase sequence centered around amino acids 60, 100, and 370 were conspicuously different relative to the tobacco sesquiterpene cyclase. The sequence similarity between the tobacco and H. muticus enzymes is suggested to be reflective of the conservation of several partial reactions common to both enzymes, and the differences may be reflective of a partial reaction unique to each enzyme.


INTRODUCTION

Terpene cyclases catalyze the cyclization of allylic diphosphate substrates to a surprising array of cyclic products. The reactions are thought to proceed through a series of partial reactions which may include an ionization of the carbon proximal to the diphosphate substituent followed by an electrophilic attack by the carbocation to a distal double bond (monocyclic formation), a second series of ionization-cyclization (bicyclic formation), rearrangements including methyl migrations, and a final stabilization due to proton elimination (1, 2, 3) . The enzymes responsible for cyclization of geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate are referred to as monoterpene, sesquiterpene, and diterpene cyclases or synthases, respectively, and represent reactions committing carbon from the central isoprenoid biosynthetic pathway to end products in the respective classes of isoprenoids.

Numerous terpene cyclases from plant and microbial sources have been partially or completely purified and characterized(4, 5, 6, 7, 8, 9, 10, 11, 12, 13) . These studies have included evaluations of the proposed reaction mechanisms (14, 15, 16, 17, 18) , efficacy of substrate analogs (19) and suicide inhibitors (20) , and the use of chemical modifying reagents to identify amino acids essential for catalysis(21, 22) . More recently, a number of fungal and plant genes for monoterpene, sesquiterpene, and diterpene cyclases have been described(23, 24, 25, 26, 27) . The plant monoterpene, sesquiterpene, and diterpene cyclases exhibit a significant degree of similarity at the amino acid level, and, at least for the sesquiterpene and diterpene cyclase, the intron-exon organization of genomic DNA is nearly identical(25, 28) . In contrast, other than perhaps the conservation of a five amino acid sequence rich in aspartate residues, very little similarity is observed between the fungal and plant sesquiterpene cyclase proteins(24, 28) . This was unexpected since the fungal enzyme, aristolochene synthase, catalyzes a reaction very similar to the plant enzyme, 5-epi-aristolochene synthase.

One implication of the sequence similarity and genomic organization observed between these plant genes is that regions of sequence conservation may correspond to functional domains, and those functional domains may mediate the catalysis of particular partial reactions common to all three enzyme reactions. Conversely, partial reactions that distinguish a cyclase reaction by virtue of its contribution to the synthesis of a unique end product may be reflected in unique amino acid sequences or domains. This inference is tested in the current work by comparing the structure of two closely related sesquiterpene cyclases from plants, 5-epi-aristolochene synthase from Nicotiana tabacum and vetispiradiene synthase from Hyoscyamus muticus (Fig. S1). Although the two enzymes catalyze overall reactions generating either an eremophilane product (bicyclic, two 6-member rings) or vetispirane product (bicyclic, one 5- and one 6-member rings), chemical rationalizations of the reaction mechanisms have suggested several partial reactions common to both and at least one final partial reaction unique to each(1, 29) . Because the N. tabacum cyclase enzyme and genes have been characterized(12, 25) , the current work was to isolate and characterize the H. muticus gene and then to compare the deduced amino acid sequences of the two cyclases for conserved and unique domains. This molecular analysis was also extended to include a comparison between other terpene cyclases.


Scheme 1: Scheme 1Proposed reaction mechanisms for eremophilane (aristolochene synthase and 5-epi-aristolochene synthase) and vetispiradiene type sesquiterpene cyclases or synthases. Partial reactions 1 and 2 are considered common to both types of cyclase and to both fungal and plant enzymes. Mechanistic differences in partial reactions 3a, 3b, and 3c are sufficient to account for the structural variants shown. Adapted from Cane (1) and Whitehead et al.(29) .




MATERIALS AND METHODS

Cell Cultures, Elicitor Treatment, and Enzyme Assays

H. muticus cell suspension cultures, initially provided by Dr. Wayne Curtis (Pennsylvania State University), were maintained in B5 media with 0.2 mg/liter 2,4-dichlorophenoxyacetic acid (30) on a weekly subculturing regime. Cultures in their rapid phase of growth, approximately 3 days after subculture and doubling in fresh weight every 24 h, were induced by the addition of a fungal elicitor preparation (100 µg of glucose equivalents per ml of cell culture) from Rhizoctonia solani. The elicitor was prepared according to Ayers et al.(31) and was kindly provided by Dr. Curtis.

Isolation and Analysis of Nucleic Acids

Total RNA was prepared by the guanidine isothiocyanate/CsCl method(32) , and poly(A) RNA was isolated by oligo(dT)-cellulose chromatography(33) . Total RNA was fractionated on 1.0% agarose gels containing formaldehyde and transferred to nylon membranes(33) . RNA blot hybridizations were performed with a 758-bp (^1)PCR probe radiolabeled using a commercial kit (Prime-It kit, Stratagene) at 60 °C in 0.25 M sodium phosphate buffer, pH 7.2, 7% SDS, 1% bovine serum albumin, 1 mM EDTA. The PCR probe was generated by amplification of H. muticus genomic DNA using oligonucleotide primers developed as part of a sequencing project of tobacco sesquiterpene cyclase genes(25) . The primers corresponded to sequences +735 to +754 in exon II (sense primer) and +1431 to +1452 in exon IV (antisense primer) of the tobacco 5-epi-aristolochene synthase genes (see GenBank accession L04680). PCR reactions were carried out under standard conditions (34) with annealing and elongation cycles of 2 min at 50 °C and 2 min at 70 °C, respectively, and the resulting 758-bp fragment was cloned into the SmaI site of pBluescript KS (Stratagene). Genomic DNA was isolated from H. muticus suspension culture cells using the cetyldimethylethylammonium bromide method(35) . Genomic DNA (5 µg) was digested with the indicated restriction enzymes, size-fractionated by electrophoresis in 0.8% (w/v) agarose gels, blotted to nylon membranes, and hybridized with radiolabeled H. muticus VS1 cDNA using identical conditions as for RNA. After hybridization, both DNA and RNA blots were washed at 45 °C, twice with 2 times SSC (1 times SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7), 0.1% SDS and twice with 0.2 times SSC, 0.01% SDS.

Construction and Screening of cDNA and Genomic Libraries

The cDNA library was constructed with poly(A) RNA isolated from 4-h elicitor-induced H. muticus cells. Preparation of double-stranded cDNA and cloning into the ZAP vector were performed according to the manufacturer's instructions (Stratagene). The genomic library was constructed by inserting EcoRI fragments (33) of partially digested H. muticus genomic DNA into the corresponding site of EMBL4 according to the manufacturer's instructions (Stratagene). Both libraries were screened using the 758-bp PCR fragment corresponding to the H. muticus vetispiradiene synthase gene spanning exon II to exon IV. DNA isolated from cDNA and genomic clones were subcloned into the appropriate restriction sites in pBluescript prior to sequencing and are designated, for example, cVS1 (cDNA vetispiradiene synthase clone 1) and gVS1 (genomic vetispiradiene synthase clone 1). Both strands of DNA were sequenced either by the dideoxynucleotide chain termination method (36) using Sequenase (U. S. Biochemical) or using an automated fluorescence-based system (Applied Biosystems). Introns were identified by comparison of cDNA sequences to that of genomic clones.

Construction of Full-length cDNAs

The largest cDNA clone isolated appeared to be missing approximately 90 nucleotides based on a comparison of the coding capacity of this cDNA to that of a tobacco sesquiterpene cyclase gene. The putative missing sequence was subsequently identified in an analysis of a genomic clone, gVS1. This genomic clone was chosen for analysis because it contained a 3`-nontranslated region identical with that found in cVS1. A reverse transcription-polymerase chain reaction (RT-PCR) strategy was subsequently designed to isolate an amino-terminal domain of a H. muticus cDNA containing a convenient HindIII site in common to that found in cVS1. The forward primer consisted of EcoRI and NcoI restriction sites (underlined), the translation initiation site (bold) and was followed by 18 bases corresponding to the coding portion of the gene (5`-GGCGAATTCCATGGCCCCAGCTATAGTGATG-3`). The reverse primer (5`-CACTTGCTTACTCAGAGG-3`) was located 577 bp downstream of the forward primer. The reverse transcription reaction was carried out in a 10-µl reaction containing 1 µg of poly(A) RNA, 25 pmol of reverse primer, 10 mM dithiothreitol, 0.5 mM each dATP, dGTP, dCTP, and dTTP, 8 units of RNase Block I (Stratagene), and first strand synthesis buffer (Stratagene). The reaction mixture was incubated for 1 h at 37 °C, followed by 5 min at 99 °C to inactivate the reverse transcriptase, and then cooled on ice for a minimum of 10 min. The PCR reaction was initiated by the addition of 40 µl of a master mix containing 10 mM Tris, 15 mM MgCl(2), 0.125 mM each dATP, dCTP, dGTP, dTTP, 25 pmol of forward primer, and 1 unit of Taq polymerase and was incubated under standard conditions (34) with annealing and elongation cycles of 2 min at 50 °C and 2 min at 70 °C, respectively. The resulting RT-PCR product of 604 bp was digested with EcoRI and HindIII, gel-purified, and ligated into the corresponding sites of pBluescript containing the partial cVS1 cDNA in a potential open-reading frame with the lacZ gene. This construct, therefore, consisted of an amino-terminal coding domain of the lacZ gene fused to a full-length H. muticus cyclase gene via an in-frame EcoRI site and was flanked at the carboxyl terminus by the 3`-noncoding region of the H. muticus cDNA. Chimeric gene 1, without the lacZ fusion, was readily isolated from this plasmid using EcoRI and KpnI, and ligated into the corresponding sites within the expression vector pGBT-T19. Chimeras 2 and 3 were constructed by ligating expression vector pGBT-T19 digested with EcoRI/KpnI and the amino-terminal portion of chimeric 1 (an EcoRI/NdeI fragment) with the corresponding carboxyl-terminal portion of either cVS2 or cVS3 (NdeI/KpnI fragments), respectively. The integrity of the chimeric genes was confirmed by DNA sequencing.

Expression Studies in Escherichia coli

Expression of the lacZ:cVS gene construct in pBluescript was examined in E. coli strain TB1. Expression of the intact, full-length chimeric cyclase genes (EcoRI/KpnI fragments of approximately 1900 bp) inserted into the bacterial expression vector pGBT-T19 (Gold Biotechnology, St. Louis, MO) was examined in TB1 host cells. Procedures for growth of the bacterial cells, induction of gene expression, and measurement of sesquiterpene cyclase enzyme activity and protein in the bacterial lysates were identical with those previously described(37) .


RESULTS

Cloning and Characterization of Vetispiradiene Synthase Genes

Earlier work demonstrated that polyclonal and monoclonal antibodies to the tobacco sesquiterpene cyclase were capable of detecting an elicitor-inducible polypeptide of 64 kDa in H. muticus which paralleled the induction of sesquiterpene cyclase enzyme activity. (^2)We therefore were encouraged to try several oligonucleotide primers used in sequencing the tobacco cyclase genes (25) in PCR reactions with H. muticus genomic DNA. Among several PCR products obtained, one dominant 758-bp product was subcloned and sequenced. In comparison to the sequence and organization of the tobacco sesquiterpene cyclase genes, the sequence of the H. muticus clone was consistent with 131 bp representing exon 2, 115 bp of intronic sequence, 371 bp complementary to exon III, 77 bp of intronic sequence, and 64 bp representing exon IV. The amino acid sequence deduced from the exonic sequence domains of the H. muticus PCR clone was approximately 75% identical with the corresponding regions of the tobacco cyclase protein. To determine if this PCR clone would be useful in isolating an elicitor-inducible sesquiterpene cyclase gene from H. muticus, RNAs isolated from control and elicitor-treated cell cultures were probed with the 758-bp fragment (Fig. 1A). No complementary transcripts were detected in RNA samples from control cell cultures, but a significant induction of mRNA was obvious within the first 2 h of treatment, reached a maximum by approximately 4 h, then declined to near control levels by 30 h. While the increase in mRNA was transient, cyclase enzyme activity was induced with a more protracted time course, reaching maximum enzyme activity 6 to 8 h after elicitor treatment and gradually declining thereafter (Fig. 1B). A very similar induction pattern and temporal relationship between the cyclase mRNA and enzyme activity was previously observed in elicitor-induced tobacco cell suspension cultures(25) .


Figure 1: Induction of vetispiradiene synthase mRNA and enzyme activity in elicitor-treated H. muticus cell suspension cultures. Cell cultures were induced with an elicitor prepared from R. solani(31) , and cell samples were collected at the indicated time points for mRNA (A) and cyclase enzyme activity determinations (B). RNA samples (5 µg) were size-fractionated by electrophoresis, transferred to nylon membranes, and then probed with a cyclase-specific 758-bp PCR product.



Three different cDNAs were subsequently isolated from a cDNA library prepared against poly(A) RNA from 4-h elicitor-treated cells and screened using the 758-bp PCR probe. cVS1 was the longest clone consisting of 1,767 bp, cVS2 was 1,095 bp long, and cVS3 was 1,295 bp long. All three clones were closely related to one another (Fig. 2) as well as to the tobacco cyclase (see below). However, the size of the protein product predicted from cVS1 was 60 kDa, 4 kDa smaller than the protein detected in immunoblots of proteins extracted from H. muticus cells, and at least 30 amino acids shorter at the amino terminus than the tobacco cyclase protein.


Figure 2: Comparison of the deduced amino acid sequences derived from 3 partial vetispiradiene synthase cDNA clones. The open diamond denotes a conserved NdeI restriction site found in all three cDNAs which was used in constructing various chimeric genes (see Fig. 4).




Figure 4: Restriction and structural maps of the H. muticus vetispiradiene synthase genomic (A) and cDNA (B) clones and chimeric genes (C). A, restriction map of a 6-kilobase pair genomic fragment harboring a complete vetispiradiene synthase gene (open box) and having a 3`-nontranslated region identical with that found in cVS1 (hatched box). Triangles below the gene represent the position of introns, and the hatched line represents the initial 758-bp PCR fragment generated. B, structural maps of the 3 different vetispiradiene synthase cDNA clones, emphasizing the sequence divergence at the 3` termini. The hatched box in cVS1 is identical with the corresponding region shown in A. C, the 3 chimeric genes were generated by first ligating a common 5` RT/PCR fragment to an overlapping HindIII site in cVS1 to generate chimeric 1, then ligating the indicated composite amino-terminal domain of chimeric 1 to the corresponding NdeI sites of cVS2 and cVS3 to generate chimeras 2 and 3, respectively.



Because efforts to isolate longer cDNAs were unsuccessful, identification of the missing amino-terminal sequence of the H. muticus cyclase protein was sought via a sequence analysis of genomic clones. This was considered somewhat problematic since 3 similar but distinctly different cDNA clones had been isolated and the longest of these hybridized to 4 to 8 fragments of H. muticus genomic DNA (Fig. 3), consistent with a small gene family of approximately 6 to 8 genes. Therefore, genomic clones harboring a cyclase gene were first isolated and then screened for their identity to one of the cDNA clones. Of the 12 positive genomic clones isolated in an initial screen, 1 clone was found to have a nucleic acid sequence identical with the 3`-nontranslated domain of the cVS1 cDNA. Additional sequence of gVS1 in the 5` direction beyond the sequence in common with the cDNA revealed a contiguous open reading frame equivalent to 35 more amino acids which were 83% identical with the amino terminus of the tobacco sesquiterpene cyclase protein.


Figure 3: DNA gel blot analysis of H. muticus genomic DNA for vetispiradiene synthase-like genes. Genomic DNA (5 µg) was digested with the indicated restriction enzymes, size-fractionated by electrophoresis, transferred to nylon membranes, and then hybridized with a radiolabeled cVS1 cDNA probe.



Based on the above information, a strategy to generate a full-length vetispiradiene synthase cDNA was developed. A RT-PCR fragment corresponding to the amino-terminal portion of the cyclase protein (from the ATG translation start site to the equivalent of amino acid 160) was ligated into cVS1 at a convenient overlapping HindIII site to generate chimeric 1 (Fig. 4). The composite gene consisted of 1665 bp coding for a 555-amino acid polypeptide with a calculated molecular weight of approximately 64.3 kDa (see Fig. 7below), similar in size to the immunodetectable H. muticus cyclase protein from elicitor-induced cells (see below).


Figure 7: A deduced amino acid sequence comparison of the N. tabacum 5-epi-aristolochene synthase and H. muticus vetispiradiene synthase (chimeric 1) proteins. Identity is denoted as dashes, differences are noted in small letters, and gaps are noted as underlining. A putative substrate binding site (DDXXD) is underlined beginning at amino acid 301, and intron positions are indicated by open triangles.



Bacterial Expression Studies

Functional confirmation of the identity of the cyclase genes was sought in expression studies. Chimeric 1 was initially inserted into pBluescript SK in-frame with 38 amino acids of the lacZ gene fused to the putative amino terminus of the cyclase gene. The predicted 68- to 70-kDa fusion protein generated from the expression of this chimeric gene was immunodetectable in the soluble fraction of Escherichia coli extracts, and while low, sesquiterpene cyclase enzyme activity was easily measurable in the extracts (Fig. 5, lane 2). No immunodetectable protein or enzyme activity was detectable in extracts from vector-control cells (lanes 1 and 3). Much higher levels of expression were observed when the chimeric gene was inserted into the expression plasmid pGBT-T19 without the lacZ fusion peptide, and the immunodetectable protein was identical in size with that found in planta (compare lanes 4 and 5). The smaller, immunodetectable peptide fragments seen in lanes 2 and 4 of Fig. 5were not visible in the vector control lanes (lanes 1 and 3) and likely represent breakdown products of the cyclase protein.


Figure 5: Expression of a H. muticus sesquiterpene cyclase gene in E. coli. Bacteria harboring various plasmid constructs were induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 3 h at 37 °C before preparing soluble protein extracts. Aliquots were used for immunodetection of the cyclase protein (A) and determination of the in vitro cyclase enzyme activity (B). M, molecular mass standards (kDa); lane 1, pBluescript SK vector in E. coli strain TB1; lane 2, pBluescript SK vector containing chimeric 1 DNA fused to the lacZ gene in E. coli strain TB1; lane 3, pGBT-T19 vector in strain TB1; lane 4, pGBT-T19 vector containing chimeric 1 DNA in strain TB1; lane 5, vetispiradiene synthase extracted from H. muticus cells.



Previous reports have documented that solanaceous plants are capable of producing a range of sesquiterpenes which raises an important question concerning the biosynthetic origins of these metabolites, especially whether they are derived from modifications to one cyclase reaction product or perhaps are derived from separate reaction products generated by different cyclase enzymes. The alignments of the deduced amino acid sequences from cVS2 and cVS3 to cVS1 indicate that they are very similar proteins and likely to have identical enzymatic functions (Fig. 2). However, to confirm a functional activity for each of the vetispiradiene synthase cDNAs, cVS2 and cVS3 were fused to an amino-terminal domain of chimeric 1 at a common NdeI site (Fig. 4), and cyclase activity in extracts of bacteria expressing the respective chimeric genes was measured. The reaction products generated from assays of bacterial extracts were identical with that generated by an extract from H. muticus cells as determined by argentation-TLC (Fig. 6) and were easily separated from aristolochene, the bicyclic sesquiterpene generated by the tobacco cyclase gene expressed in bacteria(37) . Quantification of the radioactivity in each spot indicated that >93% of the radioactivity applied migrated as a single component.


Figure 6: Comparison of the vetispiradiene synthase reaction products generated by native and recombinant forms of the enzyme by argentation-TLC. Lane 1, reaction product of native sesquiterpene cyclase extracted from H. muticus cells; lanes 2-4, reaction products from in vitro assays using extracts of bacteria (strain TB1) expressing chimeric 1, chimeric 2, or chimeric 3 DNAs inserted into the pGBT-T19 vector, respectively; and lane 5, reaction product from an in vitro assay using an extract of bacteria (strain BL21(DE3)) expressing a 5-epi-aristolochene synthase gene inserted into a pET11d vector(37) . Radioactivity in each spot was determined by liquid scintillation counting of the isolated spots.



Sequence Comparison to 5-epi-Aristolochene Synthase

The sequence comparison between the deduced amino acid sequences for the vetispiradiene synthase and 5-epi-aristolochene synthase enzymes is presented in Fig. 7. The two proteins are highly conserved throughout the protein sequence with an overall amino acid identity of 77% and a similarity of 81%. There are also several regions of conspicuous differences, notably around amino acids 60, 100, and 370.


DISCUSSION

The work presented here addresses several issues revolving around the enzymology of terpene cyclases. The first is related to the observation that plant extracts often contain several members of an isoprenoid class. For example, in pathogen or elicitor-induced solanaceous plants, 3 to 5 different bicyclic sesquiterpenes are found including capsidiol, solavetivone, phytuberin, phytuberol, rishitin, and lubimin(39) . Although these sesquiterpenes are all structurally related (eudesmane class), they differ in that the rings may consist of two 6-member rings (eremophilane subclass) or one 6- and one 5-member rings (vetispirane subclass) and various degrees of hydroxylation. An outstanding question is whether these different sesquiterpenes are generated from one sesquiterpene cyclase enzyme or more than one. This is further complicated by the observation of cyclase gene families (25) . Work from the Croteau laboratory on monoterpene cyclases has provided evidence for both these possibilities. Limonene synthase catalyzes the synthesis of one predominant product (9) while pinene synthases generate multiple products(38, 40) . In the current work, we have shown that H. muticus contains a sesquiterpene cyclase gene family, that at least 3 members of this family are expressed in response to fungal elicitors, and that each of the expressed cyclase genes encodes for an enzyme which produces one dominant (>93%) enzymatic product. A very similar induction pattern and temporal relationship between the cyclase mRNA and enzyme activity(25) , complexity of a gene family(25) , and observation of a single enzyme reaction product synthesized by extracts of bacteria expressing the tobacco 5-epi-aristolochene synthase gene (37) have been reported.

The reactions proposed for tobacco and similar sesquiterpene cyclases are complex, consisting of 3 or more partial reactions (Fig. S1, adopted from Cane (1) and Whitehead et al.(29) ). The initial isomerization of farnesyl diphosphate to nerolidyl diphosphate (not shown in Fig. S1) is followed by an intramolecular electrophilic attack by carbon 10 on the distal double bond to form germacrene A, a macrocyclic intermediate (step 1). Internal ring closure and formation of the eudesmane carbonium ion constitute step 2. For the tobacco sesquiterpene cyclase, the terminal step is a hydride shift and methyl migration giving rise to 5-epi-aristolochene. In comparison to aristolochene synthase from fungi, only the final step needs to be different in the stereospecificity of the methyl migration. Somewhat surprising then was the observation of a lack of conservation between the primary structures of the plant and fungal enzymes(24, 28) . Whitehead et al.(29) and Cane (1) have also suggested that the vetispiradiene type sesquiterpenes like solavetivone and lubimin arise via a similar mechanism. The difference resides in the third partial reaction in which ring contraction would occur due to an alternative migration of an electron pair (Fig. S1). Assuming some common ancestry for the sesquiterpene cyclase genes in solanaceous plants, we predicted that 5-epi-aristolochene synthase from N. tabacum and vetispiradiene synthase from H. muticus might share regions or domains of similarity corresponding to partial reactions 1 and 2 and at least one other region responsible for the third partial reaction exhibiting a much greater difference between the two proteins.

The primary sequence of the vetispiradiene synthase protein is very similar to 5-epi-aristolochene synthase with a more or less equal distribution of amino acid substitutions and mismatches throughout. Such conservation and even dispersal of amino acid substitutions has not allowed us to readily identify domains which might correspond to particular partial reactions in common between the two enzymes. The converse is also true. Although there are three regions within the vetispiradiene synthase protein more conspicuous because of amino acid differences in comparison to 5-epi-aristolochene synthase which, if any, of these regions is likely to contribute to the unique terminal step in the overall reaction is also difficult to discern. The apparent similarity between the plant sesquiterpene cyclases can be extended to two other recently described plant cyclases (Fig. 8). Limonene synthase (26) and casbene synthase (27) are monoterpene and diterpene cyclases, respectively, which catalyze reactions sharing mechanistic similarities to the sesquiterpene cyclases (e.g. initial ionization of the diphosphate moiety and electrophilic attack to a distal double bond). The surprising degree of similarity observed between these four plant genes (27, 28) may be somewhat reconciled after considering that the intron-exon organization of the genes for at least the sesquiterpene and diterpene cyclases is also conserved. This implies that the exonic sequences might in fact represent conserved domains which serve similar or analogous functions in each enzyme. This similarity in the protein sequence and conservation of gene structure implies that some functional assay will be required, such as site-directed mutagenesis, amino acid substitution, or even some domain swapping experiments, to evaluate the contribution of domains to a cyclase reaction.


Figure 8: A schematic representation of an amino acid sequence alignment between a mint monoterpene cyclase(26) , the H. muticus vetispiradiene synthase described herein, a tobacco sesquiterpene cyclase(25) , a castor bean diterpene cyclase (27) , and a fungal sesquiterpene cyclase(24) . Sequence alignments used deduced amino acid sequences corresponding to exons or analogous regions within proteins and were performed using the MacVector software package (IBI). Solid vertical bars correspond to intron positions within the Hyoscyamus, tobacco, and castor bean genes, and the hatched bars in the mint and fungal genes delimit the corresponding protein domains used to calculate identity scores. Numbers within the boxes indicate the number of amino acids encoded by an exon (Hyoscyamus, tobacco, and castor bean only) or corresponding region of the mint and fungal proteins. Percentages refer to identity scores between the indicated domains, and H, C, and DDXXD refer to conserved histidine-, cysteine-, and aspartate-rich residues. Adapted from Mau and West (27) and Chappell(28) .




FOOTNOTES

*
This work was supported by a grant from the National Science Foundation, the Kentucky Agricultural Experiment Station, and a Korean Government Fellowship (to K. B.). This is Journal Paper 95-06-009 of the Kentucky Experiment Station. 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(TM)/EMBL Data Bank with accession number(s) U20187 [GenBank]for chimeric 1, U20188 [GenBank]for cVS1, U20189 [GenBank]for cVS2, and U20190 [GenBank]for cVS3.

§
To whom correspondence and reprint requests should be addressed. Tel.: 606-257-4624; Fax: 606-323-1952; Chappell{at}UKCC.UKY.edu.

(^1)
The abbreviations used are: bp, base pair(s); RT-PCR, reverse transcription-polymerase chain reaction.

(^2)
K. Back, C. R. De Hass, J. Chappell, and W. R. Curtis, submitted for publication.


ACKNOWLEDGEMENTS

We thank Drs. Art Hunt and Skip Waechter for their constructive suggestions concerning this work and manuscript and Dr. Robert Klein for his help with the bacterial expression studies.


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