(Received for publication, January 10, 1995)
From the
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.
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) .
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.
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--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.
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) .
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.