Archives of Biochemistry and Biophysics Vol. 379, No. 1, July 1, pp. 137–146, 2000 doi:10.1006/abbi.2000.1865, available online at http://www.idealibrary.com on Heterologous Expression and Characterization of a “Pseudomature” Form of Taxadiene Synthase Involved in Paclitaxel (Taxol) Biosynthesis and Evaluation of a Potential Intermediate and Inhibitors of the Multistep Diterpene Cyclization Reaction1 David C. Williams,* Mark R. Wildung,* Alan Qingwu Jin,† Dolan Dalal,‡ John S. Oliver,‡ Robert M. Coates,† and Rodney Croteau2 *Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340; †Department of Chemistry, University of Illinois, 600 South Matthews Avenue, Urbana, Illinois 61801; and ‡Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912 Received February 23, 2000, and in revised form April 4, 2000 with kinetics comparable to the native enzyme. In ad- The diterpene cyclase taxadiene synthase from yew dition to the major product, taxa-4(5),11(12)-diene (Taxus) species transforms geranylgeranyl diphos- (94%), this enzyme produces a small amount of the phate to taxa-4(5),11(12)-diene as the first committed isomeric taxa-4(20),11(12)-diene (ϳϳ5%), and a product step in the biosynthesis of the anti-cancer drug Taxol. tentatively identified as verticillene (ϳϳ1%). Isotopi- Taxadiene synthase is translated as a preprotein bear- cally sensitive branching experiments utilizing (4R)- 2 ing an N-terminal targeting sequence for localization [4- H1]geranylgeranyl diphosphate confirmed that the to and processing in the plastids. Overexpression of two taxadiene isomers, and a third (taxa-3(4),11(12)- the full-length preprotein in Escherichia coli and pu- diene), are derived from the same intermediate tax- rification are compromised by host codon usage, inclu- enyl C4-carbocation. These results, along with the fail- sion body formation, and association with host chap- ure of the enzyme to utilize 2,7-cyclogeranylgeranyl erones, and the preprotein is catalytically impaired. diphosphate as an alternate substrate, indicate that Since the transit peptide-mature enzyme cleavage site the reaction proceeds by initial ionization of the could not be determined directly, a series of N-termi- diphosphate ester and macrocyclization to the verti- nally truncated enzymes was created by expression of cillyl intermediate, followed by a secondary cycliza- the corresponding cDNAs from a suitable vector, and tion to the taxenyl cation and deprotonation (i.e., for- each was purified and kinetically evaluated. Deletion mation of the A-ring prior to B/C-ring closure). Two of up to 79 residues yielded functional protein; how- potential mechanism-based inhibitors were tested ever, deletion of 93 or more amino acids resulted in with recombinant taxadiene synthase but neither pro- complete elimination of activity, implying a structural vided time-dependent inactivation nor afforded more or catalytic role for the amino terminus. The than modest competitive inhibition. © 2000 Academic Press pseudomature form of taxadiene synthase having 60 Key Words: Taxol biosynthesis; paclitaxel; taxadiene amino acids deleted from the preprotein was found to synthase; Taxus; yew. be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity Taxadiene synthase from Taxus (yew) species cata- 1 This investigation was supported in part by grants CA-55254 lyzes the first committed step in the biosynthesis of the (R.C.), GM-13956 (R.M.C.), and GM-31354 (R.C.) from the National 3 Institutes of Health, by Cytoclonal Pharmaceutics, and by McIntire- anti-cancer drug Taxol and related taxane diterpe- Stennis Project 0967 from the Washington State University Agricul- tural Research Center. 2 To whom correspondence and reprint requests should be ad- 3 Paclitaxel is the generic name for Taxol, which is now a regis- dressed. Fax: (509) 335-7643. E-mail: [email protected]. tered trademark of Bristol-Meyers Squibb. Because of the far greater 0003-9861/00 $35.00 137 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 138 WILLIAMS ET AL. FIG. 1. Cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene and subsequent elaboration to Taxol. Ac, acetyl group; Bz, benzoyl group; OPP, diphosphate moiety. noids by the cyclization of the universal precursor of where proteolytic processing of the transit peptide oc- diterpenes, geranylgeranyl diphosphate, to the olefinic curs and where the mature form of the enzyme func- product (Fig. 1) (1). This enzyme catalyzes a slow, but tions in the first step of Taxol biosynthesis. The pre- apparently not rate limiting, step in the Taxol biosyn- protein forms of the terpene synthases are often suffi- thetic pathway (2). Taxadiene synthase has been iso- ciently active to permit identification of clones by lated from yew saplings (3) and cell cultures (2), and functional heterologous expression from standard clon- the enzyme exhibits properties typical of the few diter- ing vectors (7), and the taxadiene synthase was con- pene synthases from gymnosperms and angiosperms firmed by this means (6). However, in high level ex- thus far defined (4). Initial assessment of the mecha- pression systems, the presence of the transit peptide of nism of taxadiene synthase (5) indicates that the reac- the terpene synthases often contributes to the forma- tion involves the ionization and cyclization of gera- tion of inclusion bodies and can compromise purifica- nylgeranyl diphosphate to a transient verticillyl cation tion of the soluble form(s) by promoting aggregation intermediate (1), with intramolecular transfer of the and/or the tight association with host chaperones and C11 proton to C7 to initiate transannular B/C-ring other proteins (8). Additionally, the preproteins may be closure to the taxenyl cation (2), followed by deproto- catalytically impaired relative to their native counter- nation at C5 to yield the taxa-4(5),11(12)-diene prod- parts. Soluble taxadiene synthase has been overex- uct; however, few other details of the stereochemical pressed recently as a His-tagged, thioredoxin fusion of mechanism or of the structure of this novel enzyme are the preprotein (9). The enzyme is catalytically capable known. when expressed in this form; however, such preprotein A cDNA encoding the taxadiene synthase from fusions are unsuitable for detailed mechanistic and Taxus brevifolia has been obtained by a homology- crystallographic study. based PCR4 cloning method (6). Like the monoterpene Deciphering the transit peptide-mature protein synthases and other diterpene synthases (7), taxadiene cleavage junction of terpene synthase preproteins from synthase is translated as a preprotein bearing an N- sequence information alone is not possible because of terminal targeting sequence for localization to plastids the considerable variation in primary sequence of these targeting peptides (10–13), and studies with the native enzymes to define this feature directly have indicated that most are N-terminally blocked (8, 14). Moreover, familiarity of the word Taxol, we use it in this paper in lieu of paclitaxel. detailed mass spectrometric analysis of a prototype 4 Abbreviations used: DTT, dithiothreitol; EDTA, ethylenediami- terpene synthase, limonene synthase, indicated that notetraacetic acid; FPLC, fast protein liquid chromatography; GC- plastidial proteolytic processing to the native form in MS, gas chromatography-mass spectrometry; Hepes, N-(2-hydroxy- this case was quite imprecise (8). Therefore, to prepare ethyl)piperazine-NЈ-(2-ethanesulfonic acid); LSC, liquid scintillation counting; Mopso, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; a “pseudomature” form of such a terpene synthase PCR, polymerase chain reaction; SDS–PAGE, sodium dodecylsul- requires the empirical construction and testing of a fate-polyacrylamide gel electrophoresis. series of truncated versions of the target protein. In HETEROLOGOUS EXPRESSION OF TAXADIENE SYNTHASE 139 FIG. 2. Possible mechanisms for the utilization of substrate analogs by taxadiene synthase. OPP, diphosphate moiety. this paper, we describe the construction, heterologous 65 min). The unstable chlorides, contaminated by about 10% of the methylenecyclohexene elimination product, were characterized by expression and purification of a series of truncated 1 13 forms of the preprotein, report on the kinetic proper- IR, H NMR, and C NMR spectra, and used without purification. Selected NMR data (benzene-d , 400 MHz) for 2,7-cyclogeranylgera- ties, solubility and stability of these “pseudomature” 6 nyl chloride: ␦H 1.04, 1.56, 1.60 (3s, 3 ϫ 3H,3CH3), 3.83 and 3.93 forms of taxadiene synthase, and discuss the mecha- (ABq, 2H, J ϭ 11.4 CH2Cl), 5.24 (m, 2H, vinyl H); ␦c 41.2 (CH2Cl). nistic implications of several new findings revealed by Reaction of the chlorides with (Bu4N)3HP2O7 (1.08 equiv) in CH3CN studies with the recombinant enzyme. (1 ml) containing powdered 3 Å molecular sieve was carried out for 48 h at room temp. according to literature procedures (20, 21). Purification by ion exchange chromatography, selective precipita- MATERIALS AND METHODS tion, and cellulose chromatography afforded 3(16 mg, 26%) and [1,1- 2 Substrates, standards, and inhibitors. (E,E,E)-[1-3H]Gera- H2]3 (7.9 mg, 10%) as white solids, which were characterized by 1 31 nylgeranyl diphosphate (120 Ci/mol) was prepared as described pre- TLC, H, and P NMR spectra, and negative ion FAB-MS. Data for 1 ϩ ϩ viously (15). (Ϯ)-Taxa-(4)5,11(12)-diene and (Ϯ)-taxa-4(20),11(12)- 3: H NMR (D2O 0.06% (w/v) EDTA concentrated NH4OH to pH ␦ ϫ diene (16) were gifts from Robert M. Williams, Colorado State Uni- 9, 400 MHz) 0.88 (s, 3 H, CH3), 1.41 (s, 6 H, 2 CH3), 1.48 (s, 3 H, versity (Fort Collins, CO). All other biochemicals and reagents were CH3), 1.59 (s, 3 H, CH3), 1.04–1.20 (m, 2 H, CH2), 1.21–1.34 (m, 1 H), purchased from Sigma Chemical Co. or Aldrich Chemical Co., unless 1.42–1.54 (m, 1 H), 1.56–1.68 (m, 1 H), 1.68–1.94 (m, 8 H, CH2), 4.25 ϭ otherwise noted.