CHAPTER
23
Isoprenoid Antibiotics
D.E. Cane
23.1 INTRODUCTION
Isoprenoid metabolites are widely distributed across essentially all forms of life.
Excluding acyclic polyisoprenoids of the dolichol family, prenylated proteins, and
steroidal metabolites, the vast majority are produced by plants and eukaryotic
microorganisms. Among the thousands of cyclic monoterpenes, sesquiterpenes and
diterpenes are a large number that display one or another kind of biotoxic activity,
be it antiviral, antibacterial, antifungal, herbicidal, insecticidal or cytostatic (Glasby
1982).
To date, few if any systematic studies of the production of isoprenoid
antibiotics have been carried out at the biochemical or molecular genetic level. On
the other hand, the recent successful purification of a variety of sesquiterpene
cyclases, coupled with significant progress in the molecular cloning, sequencing
and expression of structural genes encoding several of these cyclases, has allowed
detailed mechanistic studies of the enzymic formation of parent sesquiterpenes from
the universal acyclic precursor farnesyl diphosphate, as well as new insights into
the regulation of cyclase expression. Detailed investigations of several key
enzymes of isoprenoid chain elongation have complemented these studies, and have
resulted in the cloning of genes for isopentenyl diphosphate isomerase, farnesyl
633
634 Isoprenoid Antibiotics
diphosphate synthase and squalene synthase. These three enzymes catalyze key
reactions of the central isoprenoid metabolic pathway.
In this central pathway, isopentenyl diphosphate (IPP) is isomerized to
dimethylallyl diphosphate (DMAPP), and the two diphosphate esters then serve as
building blocks for the acyclic C10, C15, and C2o intermediates geranyl, farnesyl,
and geranylgeranyl diphosphate (GPP, FPP, and GGPP), respectively (Figure 231). Head-to-head reductive coupling of two FPP units yields squalene, the
universal precursor of steroids and all other triterpenes, while internal electrophilic
cyclization of FPP generates some 200 cyclic sesquiterpenes. Although to date the
genes for IPP isomerase, FPP synthase and squalene synthase have been isolated
only from yeast or mammalian cells, the corresponding genes in antibioticproducing organisms are expected to resemble them closely. The particular
relevance of isoprenoid chain-elongation enzymes lies in their close mechanistic
similarity to the terpenoid cyclases; there is also interest in factors that control the
flux of intermediates into and out of the central isoprenoid biosynthetic pathway.
GPP
Monoterpenes
FPP
Sesquiterpenes
Squalene
Figure 23-1 Central isoprenoid chain elongation pathway with branching to cyclic
terpenes and squalene.
This chapter highlights recent work from several laboratories on the
enzymes mediating key steps in isoprenoid chain elongation; it also reviews
progress in our own and other laboratories on the isolation of purified sesquiterpene cyclases, the cloning and expression of the relevant structural genes, and the
in vivo regulation of cyclase expression. No attempt is made to cover the closely
related and important topic of monoterpene cyclase enzymology, which has recently
been reviewed in depth (Croteau 1987), nor to cover recent advances in our
understanding of monoterpene cyclase expression (Gijzen et al. 1991; Lewinsohn
et al. 1991a, 1991b). Finally, little is said about the extensive mechanistic
23.2 Isoprenoid Chain Elongation Enzymes 635
investigations of enzyme-catalyzed isoprenoid cyclizations reported in the last
several years. Interested readers are referred to earlier, more comprehensive
monographs on isoprenoid biosynthesis (Porter and Spurgeon 1981), isoprenoid
enzymology (Law and Rilling 1985), and the enzymic formation of sesquiterpenes
(Cane 1990a, 1990b).
23.2 ISOPRENOID CHAIN ELONGATION ENZYMES
23.2.1 Isopentenyl Diphosphate Isomerase
Isopentenyl diphosphate: dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the reversible prototropic isomerization of isopentenyl diphosphate (IPP) to
dimethylallyl diphosphate (DMAPP), the universal electrophilic primer for the
prenyl transferase-catalyzed reactions of isoprenoid chain elongation (Figure 23-2).
Extensive mechanistic studies have shown that isomerization takes place by the net
1,3-antarafacial (anti) electrophilic allylic addition-elimination (SE0 process in
which a proton is added to the terminus of the C-3,4 double bond of IPP and the
H-2re proton is then removed, presumably by a two-base mechanism (Poulter and
Rilling 1981a). No covalent intermediates have been detected; studies with substrate analogues and a variety of inhibitors have implicated a carbocation intermediate (Reardon and Abeles 1986; Muehlbacher and Poulter 1988).
H+
OPP
IPP
Figure 23-2 Isomerase-catalyzed conversion of IPP to DMAPP.
Although the ubiquitous IPP isomerase has been isolated and purified from
a variety of sources, low enzyme titres have hampered further mechanistic
investigations. Poulter and his collaborators developed an improved six-step
procedure giving nearly 3000-fold purification of the Saccharomyces cerevisiae
enzyme (Anderson et al. 1989a). Based on the sequence of the first 35 amino acids
of the homogenous protein, two nonoverlapping oligonucleotide probes were
designed, one a 17-mer consisting of a mixture of all possible amino acid codons,
and the other a unique 56-mer based on yeast codon preferences. Both probes
were used to screen a λ library of yeast genomic DNA and to identify a 3.5-kb
insert in the DNA of five hybridizing plaques. Southern analysis of restriction
636 Isoprenoid Antibiotics
fragments and subsequent sequencing located the structural genes for IPP isomerase
in an open reading frame (ORF) of 864 bp, corresponding to a 33.5 kDa protein.
(GenBank™ accession number J05090). A long (dAdT) region upstream of the
ATG start codon was presumed to be a yeast promoter element. Although the
deduced size of the enzyme was significantly smaller than the 39-40 kDa previously
suggested by SDS-PAGE analysis of the native protein, no evidence for post-translational modification of the protein could be found. The assignment of the ORF
was supported by cloning the 3.5-kb EcoRl fragment in both orientations in a yeast
shuttle vector, YRpl7. Yeast transformed with the recombinant vector showed five
to six-fold increases in IPP isomerase activity.
Overexpression of the IPP isomerase gene was accomplished by first using
the polymerase chain reaction (PCR) to introduce Ndel and Sail restriction sites at
the ATG start site and the downstream end of the gene, respectively (Street and
Poulter 1990). The resulting 0.87-kb insert was cloned in the proper reading frame
in the£. coli expression vector pARC306N, which contained a nalidixic acid-inducible rec A promoter and the phage T7 gene-/0 ribosome binding site and leader
sequence for high level expression and translational efficiency. Transformants
produced IPP isomerase as 30-35% of their total protein, a nearly 100,000-fold
increase over the untransformed host. The 33.35-kDa recombinant enzyme had the
same mobility on SDS-PAGE gels as the native protein.
Incubation of purified recombinant isomerase with the active site-directed
irreversible inhibitor [4-3H]-3-(fluoromethyl)-3-butenyl diphosphate yielded an
inactive enzyme-inhibitor complex. The site of covalent attachment was shown to
be Cys-139 by microsequencing of the labelled peptide obtained by tryptic digestion of the inactivated protein. Further support for the implied catalytic importance
of Cys-139 has been obtained by using PCR to introduce two site-directed IPP
isomerase mutations. Strains containing the mutations C139A and C139V yielded
catalytically inactive enzyme (Street et al. 1991).
Poulter's group has also used recombinant isomerase to carry out a mechanistic study with results that have intriguing implications for understanding substrate-product binding (Street et al. 1990). Incubation of IPP with 0.1 mg of
cloned IPP isomerase in D 2 0 caused the expected rapid exchange of the H-4 and
H-2re protons of IPP and DMAPP, as monitored by proton NMR. However, an
additional but slower exchange was observed for the H-5 and H-2si protons. The
result is explained by postulating that from time to time IPP isomerase binds
DMAPP in an anomalous orientation, with the Z-methyl group (C-5) in the normal
C-4 binding pocket (Figure 23-3). Addition of a proton (deuterium) to the si face
of the anomalously oriented DMAPP double bond, and subsequent loss of a C-5
proton generates IPP that equilibrates normally with D 2 0, but now with exchange
of the original H-5 protons. Although there might be occasional misbinding of the
substrate, the actual allylic addition-elimination process would be expected to
remain stereospecific. The consequences of this anomalous binding are normally
transparent, since only the natural substrates IPP and DMAPP are generated at the
23.2 Isoprenoid Chain Elongation Enzymes 637
active site of the isomerase. Of course, such a study would not have been feasible
without adequate quantities of enzyme available as recombinant protein.
D+
.H A
"*-!
HB
N)PP
IPP
^*
DN
^
T^v
^y ^
.HA
T>PP
DMAPP
Figure 23-3 Anomalous binding and interconversion of IPP to DMAPP in the
isomerase active site.
23.2.2 Farnesyl Diphosphate Synthase
FPP synthase (EC 2.5.1.1) catalyzes a reaction (Figure 23-4) in which IPP
condenses with DMAPP and then GPP to give the acyclic diphosphate ester FPP
(Poulter and Rilling 1981a). The electrophilic allylic addition-elimination (SE>)
reaction catalyzed by FPP synthase bears a mechanistic similarity to the IPP
isomerase reaction, but involves a net suprafacial (syn) attack at C-4 of the IPP
double bond with loss of H-2re from the resultant intermediate carbocation. FPP
serves through the intermediacy of squalene as the precursor of steroids and
triterpenes, and is also the precursor of longer chain polyisoprenoids involved in
the formation of diterpenes, ubiquinones, dolichols and numerous other derivatives.
The recent discovery that prenylation of proteins is a widely occurring and
important process has stimulated a burst of interest in this phenomenon (Hancock
et al. 1989; Schafer et al. 1989; Rilling et al. 1989; Goodman et al. 1990). In
addition, FPP serves as the acyclic precursor of numerous cyclic sesquiterpenes.
Although most studies of FPP synthase have involved cholesterogenic
organisms, it is generally accepted that the biochemical details of FPP formation
are likely to be similar in all organisms. FPP synthases have been purified from
a wide variety of microbial, plant and animal sources, and to date all are
homodimers of subunitMr 38-43,000, requiring the divalent cations Mg2+ or Mn2*
for activity. Four independent groups have reported the cloning and sequencing
of genes encoding FPP synthase. Edwards and his collaborators used differential
hybridization to isolate a cholesterol-repressible rat liver cDNA that hybridized to
a specific 1.2-kb mRNA (Clarke et al. 1987). Sequencing (GenBank ™ accession
number M34477) revealed an ORF for which the predicted protein subunitMr and
amino acid sequence showed significant similarities to known chicken, pig and
human prenyl transferases, including 57% identity with a 30-amino acid sequence
implicated as an active-site peptide of the avian liver enzyme (Brems et al. 1981).
That the ORF was an FPP synthase gene was confirmed when a derived fusion
protein gave antibodies that inhibited rat liver prenyl transferase, and cross-reacted
638 Isoprenoid Antibiotics
with specific antisera to the chicken liver enzyme (Ashby and Edwards 1989).
After also investigating FPP synthase transcription in rat testes, the Edwards group
concluded that liver and testis transcripts are derived from the same gene, but are
under the control of distinct tissue-specific promoters (Teruya et al. 1990, 1991).
OPP
GPP
FPP
Figure 23-4 Formation of FPP from DMAPP and IPP.
The Edwards group has also isolated partial cDNAs for human FPP
synthase by using the rat liver cDNA to probe both HepG2 and placental cDNA
libraries (Wilkin et al. 1990). Sequencing confirmed the close similarity to the rat
liver gene (81 and 85% identity at the respective nucleotide and amino acid levels,
with increasing divergence at the 3'-end of the cDNA), and indicated that the ATG
start codon and the next seven codons were absent in the largest cDNA. The
sequence at the 5'-end of the human FPP synthase gene was established by using
anchored PCR, with the mRNA from induced cells serving as the template. The
complete sequence (GenBank™ accession number J05262) was found in an ORF
of 1138 bp, corresponding to 353 amino acids in a 40.52-kDa protein. In the
meantime, from a human fetal liver cDNA library using rat liver cDNA clones for
FPP synthase as probes, Sheares and collaborators at Merck, Sharp and Dohme
Research Laboratories had independently isolated essentially the same partial cDNA
(GenBank™ accession number M29863) lacking the first seven codons (Sheares et
al. 1989). Insertion of the cDNA into the£coRI site of pUC18 allowed expression
in E. coli of a 40-kDa β-galactosidase fusion protein that cross-reacted with
antibodies to chicken liver FPP synthase. The chimeric protein was enzymically
active and has been purified (Ding et al. 1991). Its KM values for IPP and GPP as
well as its Vmax for FPP formation were essentially identical to those of native
23.2 Isoprenoid Chain Elongation Enzymes 639
human FPP synthase.
In a separate series of experiments, Poulter's group cloned and expressed
the FPP synthase gene from S. cerevisiae (Anderson et al. 1989b). They used a
33-mer oligonucleotide coding for 11 amino acids near the N-terminus of yeast
FPP synthase as a hybridization probe to screen a pBR322 library of sized EcoRl
fragments of yeast genomic DNA. Codon assignments in the probe were based on
yeast codon usage for highly expressed genes. The screening procedure identified
a plasmid harboring a 4.5-kb EcoRl insert. The FPP synthase gene was contained
in a 1059-bp ORF encoding 352 amino acids (40.48 kDa) with 44% sequence identity to the rat liver enzyme, and 66% similarity based on conservative amino acid
substitutions (GenBank™ accession number J05091). Subcloning of the EcoRl
fragment into a yeast shuttle vector led to 2 to 3-fold overexpression of FPP
synthase activity in transformed yeast cells. Further experimentation based on gene
disruption and Southern hybridization with separated yeast chromosomes established that the FPP synthase gene resided as a single copy on chromosome XI.
An alternative approach was used by Fujisaki et al. (1990) to clone and
sequence ispA from E. coli. This gene, which encodes FPP synthase activity, had
been mapped at minute 10, between the tsx and Ion loci, on the E. coli chromosome (Fujisaki et al. 1989). Various phage X2H5 restriction fragments known to
contain DNA proximal to the Ion gene (Kohara et al. 1987) were subcloned into
pUC19, and the recombinant plasmids were used to complement a host containing
a temperature-sensitive ispA mutation. Further restriction digests and sequencing
identified an 897-bp ORF encoding a 32.16-kDa protein (GenBank™ accession
number D00694). The E. coli ispA gene product showed limited amino acid sequence identity with eucaryotic FPP synthases, except in the aspartate-rich terminal
region which contained the sequence VQDDILD, in common with several other enzymes of this class (see below). When transformed with a plasmid carrying the
entire 899-bp ORF downstream of the lac promoter, both ispA* and ispA" E. coli
hosts had elevated FPP synthase activity, and a protein of the expected size was
generated in maxicells harboring plasmids with the ispA coding sequence.
In related studies, Ashby and Edwards (1990) characterized the structural
gene encoding hexaprenyl diphosphate (HPP) synthase in yeast. This prenyl transferase, which is required for isoprenoid chain elongation in coenzyme Q biosynthesis, catalyzes the stepwise 1-4' condensation of allylic diphosphates with IPP by
a mechanism essentially identical to the prototype FPP synthase reaction. The HPP
synthase gene is a 1419-bp ORF encoding 473 amino acids in a 52.56-kDa protein
subunit (GenBank™ accession number J05547). The amino acid sequences of yeast
HPP synthase and rat FPP synthase had similar hydrophobicity profiles. Even
more interesting were the two aspartate-rich domains of HPP synthase, and the
presence of a consensus sequence, (I,L, or V) XDDXXD, found in rat, human and
yeast (as well as E. coli) FPP synthases. These domains are tentatively implicated
in substrate binding, possibly by formation of salt bridges to the divalent metal of
allyl or homoallyl diphosphate Mg2+ complexes. Unfortunately, the generality of
640 Isoprenoid Antibiotics
this observation is uncertain since the domains are absent from yeast IPP
isomerase. Site-directed mutagenesis has been used to show that replacement of
the third aspartate by glutamate in rat FPP synthase had no significant effect on the
prenyl transferase activity observed, while substitution of glutamate for the first
aspartate reduced Vmax 90-fold and increased the KM for IPP without affecting the
KM for GPP Marrero et al. 1992). A third domain in both HPP and FPP synthases
comprises the same arginine- and lysine-rich motif previously implicated in the
active-site peptide of FPP synthase.
23.2.3 Squalene Synthase
Squalene (Figure 23-5; structure 1) is the universal precursor of steroids and
triterpenes. The conversion of FPP to squalene proceeds initially by electrophilic
addition of FPP to the 2,3-double bond of a second equivalent of FPP, followed
by cyclization and deprotonation to generate the cyclopropylcarbinyl intermediate
presqualene diphosphate (PSPP) (Poulter and Rilling 1981b). Ionization of PSPP
followed by rearrangement and reduction with NADPH then gives squalene. While
Figure 23-5 Formation of squalene (structure 1) from two equivalents of FPP via
presqualene diphosphate (PSPP).
this reaction is not directly involved in the formation of sesquiterpenes, it is
nevertheless of considerable relevance because of the FPP binding sites in the
enzyme and its mechanistic similarities to several features of terpenoid cyclase
reactions.
Several groups have independently cloned squalene synthase genes. At
23.2 Isoprenoid Chain Elongation Enzymes 641
Bristol-Myers Squibb, Jennings et al. (1991) screened a yeast genomic library for
transformants that complemented squalene synthase-deficient (erg9) mutants. The
ERG9 gene was found in an ORF of 1332 bp, corresponding to a 51.75-kDa
protein of 444 amino acids (GenBank™ accession number M63979; see also
Fegueur et al. 1991). Analysis of the sequence revealed a weak homology
(KTNIIRD) to the aspartate-rich motif previously observed in FPP synthases and
presumed to be involved in allylic diphosphate binding. Analogous sequences are
present in two closely related enzymes implicated in carotenoid biosynthesis. The
crtB gene of Rhodobacter capsulatus encodes an enzyme catalyzing the conversion
of GGPP to prephytoene diphosphate and containing the sequence VADDEVD,
while crtE from the same organism (EMBL accession number X52291) encodes
phytoene synthase containing the sequence IADDLKD (Armstrong et al. 1989).
The corresponding crtB gene of Erwinia herbicola, on the other hand, has the
slightly modified sequence HCDDVID (GenBank™ accession number M38423)
(Armstrong et al. 1990).
Hydrophobicity plots of yeast squalene synthase predicted a number of
putative transmembrane sequences, and were consistent with the fact that squalene
synthase is a membrane-bound protein. The identity of the ORF was confirmed
by replacing portions with HIS3 or LEU2 DNA and using the resulting plasmid
constructs to disrupt the chromosomal ERG9 allele of recipient yeast strains by
homologous recombination. Squalene synthase was also expressed in E. coli by
inserting a 1.5-kb BglU PCR fragment containing the ERG9 coding sequence plus
12 bp of upstream untranslated sequence into pTTQ8 containing the tac promoter.
The E. coli transformants showed squalene synthase activity comparable to that of
wild-type yeast, but examination of cell extracts indicated that much of the protein
was present in inclusion bodies. More recently, a truncated version of the yeast
squalene synthase gene has been expressed in E. coli, and accounted for 2-5% of
the soluble protein (Zhang et al. 1993).
Using an alternative approach, Händler et al. (1991) at Sandoz screened a
yeast genomic library with mixed oligonucleotide probes based on the known Nterminal amino acid sequence of squalene synthase; they isolated a clone containing
320 bp of coding sequence. By using this clone to rescreen the library, the
complete yeast squalene synthase gene was found. After truncation to remove the
sequence encoding the 24 C-terminal amino acids that presumably corresponded to
the —3 kDa membrane anchor, the gene was cloned in E. coli using the pET3a
(Novagen) expression vector under the control of the T7 promoter; enzymically
active squalene synthase was produced as 20% of the soluble protein LoGrasso et
al. 1993). Also very recently, Schechter's group (McKenzie et al. 1992) has
described the cloning and expression of the cDNA for rat hepatic squalene synthase
(GenBank™ accession number M95591). The sequence of this gene showed 59%
identity over 465 nucleotides with the yeast squalene synthase gene sequence, and
at least two regions, possibly of catalytic significance, showed similarity to regions
in prephytoene synthase genes.
642 Isoprenoid Antibiotics
23.3 SESQUITERPENE SYNTHASES
Cyclic sesquiterpenes are derived from FPP by ionization of the allylic diphosphate
and electrophilic attack of the resultant allylic cation on either the central or distal
double bond of the substrate, followed by well-precedented cationic transformations
that culminate in quenching of the positive charge by deprotonation or capture of
an external nucleophile such as water (Figure 23-6: Cane 1981). This process,
which can account for the formation of nearly 200 known cyclic sesquiterpenes,
is the intramolecular counterpart of the well studied prenyl transferase reaction.
The similarity is further reflected in the enzymology of those sesquiterpene synthases isolated and studied to date (Croteau and Cane 1985; Cane 1990a, 1990b).
In common with monoterpene cyclases, these enzymes are soluble proteins, either
monomers or homodimers of subunit Mr 40-60,000, requiring no cofactors other
than a divalent metal; Mg2+ is usually preferred. The turnover numbers are
modest (0.05-0.5 s"1) but significantly above the rate of metal-ion catalyzed
solvolysis; KM values for FPP are generally in the 0.5-5 μΜ range. Detailed
mechanistic studies of the stereochemical course of several of these reactions have
suggested that the folding of the acyclic substrate at the active site of the synthase
plays a major part in determining the structure of the characteristic cyclic product
eventually formed (Cane 1985).
Figure 23-6 Enzymatic cyclization of FPP to cationic intermediates leading to the
formation of cyclic sesquiterpenes
23.3 Sesquiterpene Synthases 643
In considering the role of the cyclase itself in forming cyclic sesquiterpenes, several key questions remain to be answered: (i) How is substrate folding
controlled? (ii) How is positive charge managed, and how does the cyclase avoid
annihilation by reactive carbocation intermediates? (iii) What is the structure of
the cyclase active site? Enlightenment may well come from studies on the mechanistically related isoprenoid chain-elongation enzymes. However, to address these
questions directly, we and others have pursued a variety of interrelated approaches.
These include the use of substrate and intermediate analogues as reversible and
irreversible cyclase inhibitors, the use of anomalous substrates that undergo abortive cyclizations, and the cloning, sequencing and overexpression of terpenoid synthase genes. The remainder of this section will cover progress in the latter area.
23.3.1 Trichodiene Synthase
Trichodiene synthase (Cane et al. 1985; Cane and Ha 1988) catalyzes cyclization
of FPP to trichodiene (Figure 23-7; structure 2), the parent hydrocarbon of numerous fungal mycotoxins with antibiotic, cytostatic and plant pathogenic activities
(Tamm and Breitenstein 1980; Grove 1988). Many of these metabolites are potent
inhibitors of protein biosynthesis, and their contamination of agricultural products
has posed an important public health problem, particularly in tropical regions.
Purified trichodiene synthase from Fusarium sporotrichioides is a homodimer of subunit Mr 45,000 (Hohn and vanMiddlesworth 1986). The enzyme from
Figure 23-7 Conversion of FPP to trichodiene (structure 2) showing the folding of the
substrate and likely intermediates.
644 Isoprenoid Antibiotics
Gibberellapulicaris (anamorph: Fusariwn sambucinum) has been partially purified
and characterized (Hohn and Beremand 1989a); it reacted with antibodies raised
against the F. sporotrichioides cyclase. Use of the antibodies to screen a Xgtll
genomic library of F. sporotrichioides DNA led to the isolation of a trichodiene
synthase (Tox5) gene, encoded in an 1182-bp ORF containing an unusual 60 nt inframe intron (GenBank™ accession number M27246) (Hohn and Beremand 1989b).
Excision of the intron and subcloning of the coding sequence in the E. coli expression vector pDR540 allowed bacterial expression of trichodiene synthase (Hohn and
Plattner 1989a). The transformed cells also contained detectable quantities of
trichodiene itself, due to appropriation and cyclization of endogenously generated
FPP. The level of expression of recombinant trichodiene synthase was relatively
low; based on a comparison of the observed specific enzyme activity (0.07 nmol
min"1 mg"1 of protein) with that of homogeneous native fungal enzyme (102 nmol
min"1 mg"1 of protein), it accounted for less than 0.1% of total cellular protein.
By subcloning the trichodiene synthase gene in pLMl, an E. coli expression vector
with an inducible T7 promoter and a T7 gene-10 transcription initiation sequence
and ribosome binding site (Sodeoka et al. 1993), we recently succeeded in
expressing trichodiene synthase in an amount representing about 25% of the soluble
cellular protein (Cane et al. 1993a). The recombinant enzyme appears to be
identical in all respects to the native fungal enzyme, and is much more conveniently and rapidly produced from E. coli. Although at one point there had been some
concern that the scavenging of endogenous FPP might somehow be deleterious to
the host, this does not seem a problem if expression is tightly controlled, and stable
transcripts are generated.
By using a BamW\ fragment containing the coding sequence for the F. sporotrichioides trichodiene synthase gene as a hybridization probe to screen a library
of G pulicaris genomic DNA, Hohn and Desjardins (1991) isolated the corresponding Tox5 gene of G. pulicaris. Comparison of the two sequences revealed a
significant (89%) identity at the nucleotide level, including the presence of the 60nt intron in the G. pulicaris gene (GenBank™ accession number M64348). The
aligned sequences also showed 96% identity at the amino acid level, the primary
difference being the presence near the C-terminus of the G. pulicaris enzyme of
an additional nine amino acids, for a total of 383 (44.7 kDa) compared to 374
amino acids (44 kDa) for the F. sporotrichioides cyclase. Neither gene showed
overall sequence similarity to known genes, including those coding for isoprenoid
chain-elongation enzymes. Interestingly, however, both genes contain the aspartate-rich motif previously noted in FPP synthase. The product of the F. sporotrichioides Tox5 gene has the sequence VLDDSKD starting at amino acid 98,
consistent with the (I,L or V)XDDXXD consensus sequence, while the corresponding protein of G. pulicaris has the sequence VLDDSSD starting at the same
position. In addition, both enzymes have a short sequence rich in basic amino
acids (F. sporotrichioides, DRRYR; G. pulicaris, DHRYR), similar to the
presumptive active-site peptide of avian liver FPP synthase and other prenyl
23.3 Sesquiterpene Synthases
645
transferases. So far these similar sequences are only suggestive of homology;
their significance remains to be experimentally demonstrated. Furthermore, the
relationship to the trichodiene synthases of more distantly related organisms is
uncertain, since antiserum to F. sporotrichioides trichodiene synthase did not crossreact with the enzyme from Myrotheciwn verrucaria (Hohn et al. 1991).
An 889-bp fragment of the G. pulicaris Tox5 gene, corresponding to amino
acids 29 through 303, was inserted in the fungal cloning vector pUCHl carrying
a hygromycin B resistance gene; the recombinant plasmid was used to transform
G. pulicaris and disrupt the Tox5 gene by homologous recombination (Hohn and
Desjardins 1991). The resultant Tox5~Hygr strains, which were mitotically and
meiotically stable, have been used to study the relationship between trichothecene
production and virulence in plant pathogenicity, as well as the late stages of
trichothecene biosynthesis (Desjardins et al. 1993).
Hohn and Ohlrogge (1991) have also demonstrated heterologous expression
of trichodiene synthase in a higher plant. By inserting the coding sequence of the
F. sporotrichioides Tox5 gene along with the cauliflower mosaic virus 35S promoter into the plant expression vector pBI121, it was possible to transform tobacco
(Nicotiana tabacum) leaf discs. Leaf tissue from the regenerated kanamycin-resistant plants contained trichodiene synthase activity as well as detectable quantities
of trichodiene. These experiments not only demonstrate the feasibility of expressing microbial cyclase genes in higher plants, but also open up the possibility of
generating novel plant metabolites conferring resistance to insect pests or fungal
pathogens, or other desirable properties.
23.3.2 Pentalenene Synthase
Pentalenene synthase (Cane and Tillman 1983) catalyzes cyclization of FPP to the
tricyclic sesquiterpene pentalenene (Figure 23-8; structure 3), the parent hydrocarbon of the pentalenolactone family of streptomycete antibiotics (Seto and Yonehara 1980). The presence of cyclic sesquiterpenes in a procaryotic organisms is
rare but not unique (Gerber 1971). Pentalenolactone itself acts as a site-directed,
irreversible inhibitor of glyceraldehyde-3-phosphate dehydrogenase, binding
covalently to each of four active-site cysteines of this tetrameric glycolytic enzyme
(Hartmann et al. 1978; Mann and Mecke 1979; Cane and Sohng 1989; Sohng
1991). In extensive studies of the isoprenoid origin of pentalenolactone, Cane et
al. (1979, 1981) demonstrated the conversion of labeled pentalenene to pentalenolactone by cultures of Streptomyces UC5319 (Cane and Tillman 1983; Cane et al.
1984. We have isolated a soluble cyclase from the same organism, which mediates
the Mg2+-dependent formation of pentalenene from FPP (Cane and Tillman 1983).
Maurer and Mecke (1986) examined the variation in pentalenene synthase activity
as a function of time in cultures of S. arenae TÜ 469; the activity reached a
maximum after 30-40 h of cultivation, then sharply declined. Some evidence was
also obtained for inhibition of the cyclase by the eventual endproducts pentalenolactone
646 Isoprenoid Antibiotics
Figure 23-8 Conversion of FTP to pentalenene (structure 3).
chlorohydrin or pentalenolactone 0-methyl ether, although this inhibition has not
been further characterized. Extensive labelling studies have led to a detailed
picture of the mechanism and stereochemistry of the cyclization reaction itself
(Cane et al. 1990a).
With the aim of isolating and expressing the pentalenene synthase gene, we
purified the cyclase to homogeneity from Streptomyces UC5319 and showed it to
be a monomer of MT 42,500, based on SDS-PAGE mobility (Cane and Pargellis
1987; Cane et al. 1991; Sohng 1991). Using the purified enzyme we obtained a
27-amino acid sequence for the N-terminus, as well as the sequence of three internal tryptic peptides. Based on these sequences, we designed mixed oligonucleotide
primers, taking into account preferred codon usage in the highly GC-rich Streptomyces, and used these for PCR amplification of a 450-bp segment of genomic
DNA. Sequencing indicated that the PCR product encodes the expected tryptic
peptides. By using the 450-bp PCR product as a hybridization probe to screen
both plasmid and phage libraries of streptomycete genomic DNA, the complete
pentalenene synthase gene was isolated (Cane et al. 1994). The nucleotide sequence of the cloned DNA fragment contained an ORF (GenBank™ accession number U05213) of 1014 bp encoding a protein of 337 amino acids (MT 38,002). The
ORF, defined by its characteristic streptomycete G+C bias and codon usage, was
preceded (12 nucleotides upstream) by a plausible ribosome binding site, and was
followed by a 9-bp inverted repeat presumed to be a transcription terminator. The
deduced amino acid sequence showed no significant similarity to database sequences, including those deduced from known sesquiterpene synthase and prenyl
transferase genes. On the other hand, the short aspartate-rich sequence LFDDLFD
beginning at amino acid 78 conforms to the consensus sequence (I,L,V)XDDXXD
found in prenyl transferases and in both trichodiene synthase and aristolochene syn-
23.3 Sesquiterpene Synthases 647
thase (see section 23.3.3). Pentalenene synthase has been expressed in E. coli
BL21(DE3) by using PCR to insert the coding sequence into the T7 expression
vector pLMl. The recombinant enzyme appears to be identical in every respect
to the native streptomycete cyclase.
23.3.3 Aristolochene Synthase
Aristolochene (Figure 23-9; structure 4) is a sesquiterpene hydrocarbon isolated in
one or the other antipodal form from a variety of sources that include the fungus
Aspergillus terreus (Cane et al. 1987, 1990c), the plant Aristolochia indica (Govindachari et al. 1970) and soldiers of the termite Syntermes (Baker et al. 1981).
The (+)-enantiomer is the probable precursor of PR-toxin, produced by Penicillium
roquefortii, and is a likely intermediate in the formation of a large family of related
fungal toxins and sporogens (Moreau et al. 1981; Gorst-Allman and Steyn 1982;
Chalmers et al. 1981).
Figure 23-9 Conversion of FPP to aristolochene (structure 4).
Aristolochene synthase, catalyzing the conversion of FPP to aristolochene,
has been isolated from both A. terreus (Cane et al. 1989) and P. roquefortii. The
purified P. roquefortii cyclase (Hohn and Plattner 1989b) has a subunit MT of
37,000, somewhat lower than the apparent native molecular weight of 48 kDa
deduced from gel filtration chromatography. We have reported a series of mechanistic studies of the cyclization using a sample of the A. terreus enzyme (Cane et
al. 1989, 1990b) purified 500-fold but not yet completely homogeneous (Cane and
Tsantrizos unpublished). Although the A. terreus and P. roquefortii enzymes catalyze the same reaction, the proteins differ somewhat; the former has a higher native
Mt (ca 56,000), and its KM for FPP (3 μΜ) is also higher than that of the P. roquefortii enzyme (0.5 μΜ). In preliminary experiments, crude extracts of A. terreus
648 Isoprenoid Antibiotics
did not appear to cross-react with antibodies to the purified Penicillium protein
(Cane and Oliver unpublished).
Proctor and Hohn (1993) have cloned the aristolochene synthase gene from
P. roquefortii, using partial amino sequence information to design degenerate PCR
primers which were used to amplify a 934-bp segment of genomic DNA. Use of
the PCR product to screen a λ-ΖΑΡ genomic library of P. roquefortii, led to
characterization of a 1126-bp sequence with two potential introns and a region
coding for a 39.1-kDa protein (GenBank™ accession number L05193). The amino
sequence showed no significant similarity to any known protein, including trichodiene synthase, although the sequence LIDDVLE did appear, starting at amino acid
113. In addition, the aristolochene synthase gene did not hybridize to A. terreus
genomic DNA, suggesting that the aristolochene synthases from these two only
distantly related organisms are not homologous. Using the same PCR-based
cloning strategy and the pLMl expression vector as described for both the pentalenene and aristolochene synthase genes, we have recently produced active recombinant P. roquefortii aristolochene synthase in E. coli (Cane et al. 1993b).
23.3.4 epi-Aristolochene Synthase
Numerous plants produce antifungal metabolites in response to tissue damage or
exposure to fungal elicitors. Members of the Solanaceae, which produce sesquiterpenoid phytoalexins, have been particularly well-studied. For example, the eremophilane sesquiterpene capsidiol (Figure 23-10; structure 5) is produced by several
species, including Capsicum frutescens (sweet pepper: Stoessl et al. 1972), Datura
stramonium (jimsonweed: Ward et al. 1976) and N. tabacum (tobacco: Bailey et
al. 1975). Two groups have shown that challenging tobacco cell cultures with eel
Figure 23-10 Conversion of FPP to e/;/-aristolochene (structure 6), the sesquiterpene
hydrocarbon precursor of the phytoalexin capsidiol (structure 5).
23 A What next ? 649
lulase or various fungal elicitors induces a sesquiterpene cyclase that catalyzes the
conversion of FPP to the bicyclic hydrocarbon e/?/-aristolochene (Figure 23-10;
structure 6), which in turn is a precursor of capsidiol and related metabolites
(Whitehead et al. 1988, 1989, 1990; Threlfall and Whitehead 1988; Vögeli and
Chappell 1988). The mechanism by which epz'-aristolochene is formed almost certainly resembles the mechanism of the aristolochene synthase reaction, the major
difference lying in the precise stereochemistry of FPP folding at the active site of
each cyclase. The existence of two enzymes catalyzing such closely related conversions provides an opportunity to study the relationship between protein structure
and catalysis. Vögeli et al. (1990) have purified e/?/-aristolochene synthase to
homogeneity. SDS-PAGE analysis of the apparently homogeneous protein showed
it to be a mixture of two polypeptides of 61.5 and 63.5 kDa; both reacted with
monoclonal antibodies raised to purified ^/-aristolochene synthase. The most
likely explanation at present is that the smaller component is a partial proteolysis
product, although proof of this must await isolation of the gene and a study of its
expression. It is interesting to note that the monoclonal antibodies did not crossreact with P. roquefortii aristolochene synthase (Hohn et al. 1991).
Although expression of all terpenoid cyclase activity is presumably under
some sort of intracellular control, e/?/-aristolochene synthase expression provides
a unique experimental model for the study of metabolite induction since the primary signals, if not yet the means of signal transduction, are already well known.
Vögeli and Chappell (1990) have shown that induction of φΖ-aristolochene synthase
is under transcriptional control; levels of cyclase mRNA correlated with cyclase
activity and with the content of immunoactive protein in treated and untreated cells.
In conifers, Croteau's group has carried out a series of detailed studies on the
relationship between tissue wounding and monoterpene cyclase activities, the
products of which play an important role in the defense of these plants against
insect predators (Gijzen et al. 1991; Lewinsohn et al. 1991a, 1991b).
Facchini and Chappell (1992) have recently isolated two full-length epiaristolochene synthase genes that mapped 5 kb apart on one cloned 15-kb genomic
DNA fragment. From 388 bp upstream of the transcription initiation site to 40 bp
downstream of the stop codon, the two sequences were identical (GenBank accession number L04680). The 1479 bp ORF in each contained five introns, and
encoded a 56,828 Da protein. The aspartate-rich cyclase motif appeared twice, as
HADDILE and IVDDTFD. The deduced N-terminal amino acid sequence was
identical to that determined for the native tobacco protein. Interestingly, hybridization with genomic DNA of N. tabacum detected more than a dozen such
sequences.
23.4 WHAT NEXT ?
Impressive progress has already been made in purifying terpenoid metabolic enzymes, and in cloning, sequencing and expressing the relevant structural
650 Isoprenoid Antibiotics
genes. There is bound to be continued, rapid progress in these and related areas.
For example, Croteau's group (Colby et al. 1993) has recently cloned, sequenced
and expressed in E. coli the cDNA for a monoterpene cyclase, 4-(5)-limonene synthase, produced in the oil glands of spearmint {Mentha spicata). The ORF for the
synthase contained 1800 bp (GenBank™ accession number L13459). The deduced
amino acid sequence contained a putative plastidial transit peptide of ~ 89 amino
acids, and a mature protein of —510 amino acids. The estimated size of 59.8 kDa
is within 10% of the Mr measured for the native protein by gel permeation chromatography and polyacrylamide gel electrophoresis. Because the N-terminus of the
native limonene synthase is blocked, and has not been sequenced, the cleavage site
for the transit peptide is not yet known. Two aspartate-rich signatures, VIDDIYD
at amino acids 350-356 and VDDTSYD at amino acids 397-403, were present.
Remarkably, although limonene synthase showed no sequence similarity to known
microbial cyclases, a comparison with other higher plant cyclases revealed 34%
identity and 56% similarity to ep/-aristolochene synthase, the sesquiterpene cyclase
from tobacco, and 31% identity and 53% similarity to casbene synthase, a diterpene cyclase from castor bean (C.J.D. Mau and C.A. West unpublished).
In spite of the progress, much terpenoid enzymology and genetics remains
unknown. Isolation of the genes for isoprenoid chain elongation and of the structural genes for sesquiterpene cyclases has in most instances required preliminary
purification to homogeneity of the relevant enzyme, so that oligonucleotide probes
or antibodies could be prepared. In one case, DNA coding for F. sporotrichioides
trichodiene synthase was used successfully to screen for the Tox5 gene in the closely related G. pulicaris, but the apparent lack of homology between cyclases catalyzing identical reactions in distantly related organisms is likely to limit the utility
of heterologous DNA probes, or immunoblotting methods relying on cross-reactivity. On the other hand, the recent observation of strong sequence identities among
cyclase genes, both within single plant species and among genes from diverse families, suggests that hybridization may turn out to be an effective strategy for
isolating plant-derived cyclase genes.
Although a few genes involved in isoprenoid chain elongation have been
isolated by complementation of blocked mutants, it is unlikely that the laborintensive screening methods required for selection of the necessary cyclase mutants
will be widely applied; the absence of cyclase activity ordinarily has no deleterious
effect on the survival of laboratory cultures, in contrast to the effects of blocking
the central isoprenoid biosynthetic pathway. Therefore, in the near future at least,
isolation of additional sesquiterpene biosynthetic genes will probably still require
the painstaking purification of relevant cyclases.
The recent successful use of a mechanism-based inactivator to covalently
modify limonene synthase (Croteau et al. 1993), and the observation that this same
inactivator is effective against a wide range of monoterpene synthases, suggests the
possibility of purifying cyclases that have been covalently labeled with radioactive
inactivator. This would provide a powerful and rapid method for purifying and
References
651
sequencing the numerous monoterpene synthases, and thereby gaining access to
their genes.
The successful expression of trichodiene synthase in both E. coli and N.
tabacum suggests that heterologous expression of cyclase activity will not in itself
be a major problem, and may present exciting opportunities for the development
of transgenic plants with desirable metabolic properties or resistance to microbial
and insect pests. The recent overexpression of trichodiene synthase, pentalenene
synthase and aristolochene synthase has now provided the quantities of protein
needed for studies of single turnover kinetics, identification of active site residues,
and X-ray analysis of single crystals with and without bound inhibitors.
The presence of a common aspartate-rich motif in numerous prenyl transferases as well as in all cyclases isolated to date, coupled with the effect of sitedirected mutations in the aspartate residues on prenyl transferase activity, implicates this motif in the catalysis of cyclization reactions. Whether the role of the
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role of the arginine-rich sequence present in both trichodiene and pentalenene
synthases is still unclear, although site-directed mutagenesis recently carried out on
trichodiene synthase by us in collaboration with Dr. Thomas Hohn has established
the importance of this domain to both binding and cyclization of farnesyl diphosphate (D. Cane and T. Hohn unpublished). The next several years should witness
significant advances in our understanding of the detailed mechanisms of molecular
recognition and catalysis by these versatile and fascinating enzymes.
ACKNOWLEDGEMENTS
Portions of the work reviewed here were carried out in my laboratory and
were supported by grants GM2217 and GM30301 from the National Institutes of
Health. I thank Dr. Thomas Hohn of the U.S. Department of Agriculture, Peoria,
IL, for providing access to results in advance of publication, and for reading part
of the manuscript.
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