Proc. Nadl. Acad. Sci. USAVol. 91, pp. 7370-7374, July 1994Biochemistry

Isolation and characterization of the gene encoding 2,3-oxidosqualene-lanosterol cyclase from Saccharomyces cerevisae

(sterd boynthesb/gt complemenItaI/catoi terns)

ZHENG SHI, CHRISTOPHER J. BUNTEL, AND JOHN H. GRIFFIN*Department of Chemistry, Stanford University, Stanford, CA 94305-5080

Communicated by William S. Johnson, April 11, 1994 (received for review February 3, 1994)

ABSTRACT TheERG7geneencodingoxidosqualene-lans-terol cyclase [(S)-2,3-epoxysqualene mutase (cyclzn, lanos-tl fig), EC 5.4.99.7 from Saccharomyces cerevuiae hasbeen cloned by genetic entato of a cy dd etcK7 strain. TheDNA sequence ofthis gene has been detenninedand found tocontain anopen readlngframe of2196 nt ( ncudingstop codon) that encodes a prd protein of731 amino acids.The predicted molecular mass of the S. cerevasiae cyclase, 83.4kDa, is similar to the pre d moeulr masses of the oxido-squalene-lanosterol cyclase from Candida aicans and the oil-dosqualene-cycloartendo cyclase from Arabidopsis Thaia, aswell as to the molecular masses aid to vertebrate oxido-squalene-lanosterol cyclases; however, it is subtantially largerthan the molecular mass aid to purified S. cerevisaecyclase. At the level ofDNA and predicted amino acd sequences,the S. cerevwiae and C. alicans cydases share 56% and 63%identity, respectively. Tryptophan and tyrosin residues areunusually abundant in the predicted amino add sequences of(oildo)-squale cyclases, leading to a hypothesis that electron-rich aromatic side chains from these residues are essentialfeatures of cyclase active sites.

Oxidosqualene cyclase enzymes (EC 5.4.99.) catalyze anarray of complex cyclization and cyclization/rearrangementreactions that have captivated scientists since the elucidationof the structure of cholesterol (1-5). As the initial step in astructure/function analysis of this family of biocatalysts, werecently determined and reported the DNA sequence of anoxidosqualene cyclase, the oxidosqualene-lanosterol cyclasefrom the fungus Candida albicans (6). The gene encoding thisenzyme was cloned by Kelly et aL (7) on the basis of its abilityto complement cyclase-deficient (erg7) strains of Saccharo-myces cerevisiae. The DNA sequence of the C. albicanscyclase gene predicts a protein of 728 amino acids withmolecular mass of 83.7 kDa. This size is similar to the relativeMr values reported for purified vertebrate oxidosqualene-lanosterol cyclases (73,000-78,000) (8), which also producelanosterol (Fig. 1) and to the predicted molecular mass for theoxidosqualene-cycloartenol cyclase from Arabidopsisthaliana (86 kDa) (9). However, the predicted molecular massfor the C. albicans cyclase is substantially larger than the26,000 Mr reported for the cyclase purified from another yeastspecies, S. cerevisiae (10). This discrepancy and the desire tolearn more about the S. cerevisiae enzyme, which has playeda central role in the development of current understandingabout the molecular details ofcyclization/rearrangement (11),prompted us to isolate and characterize the S. cerevisiaecyclase gene. In this paper, we report the cloning oftheERG7gene encoding the S. cerevisiae oxidosqualene-lanosterol cy-clase using genetic complementation. The DNA and derivedamino acid sequences ofthis gene bear great similarity to those

Oxidosqualene- 1

I|0 LanosterolCyclase

HO0

(3S).2,3-Oxldosqualene Lanosterol

FIG. 1. Conversion of (3S)-2,3-oxidosqualene to lanosterol cat-alyzed by oxidosqualene-lanosterol cyclase.

of the C. albicans cyclase and predict that the S. cerevisiaecyclase is a protein of 731 amino acids with a molecular massof 83.4 kDa. The predicted amino acid sequence of this andother cloned cyclase enzymes is unusually rich in tryptophanand tyrosine residues, and we propose that electron-richaromatic side chains are essential features of cyclase activesites.

EXPERIMENTAL PROCEDURESStrains, Media, and DNA Manipulations. S. cerevisiae

strain SGL9 (erg7 ura3-52 hem3-6 gal2) is a segregant of across between strains GL7 (erg7-s hem3-6 gal2) (12) and 9a(ura3-52), obtained from John McCusker (Department ofBiochemistry, Stanford University). Strain SGL9 was grownat 30TC on medium supplemented with ergosterol (0.001%),Tween 80 (0.5%), and hemin hydrochloride (0.00015%) (12).The genomic library of S. cerevisiae in vector AYES wasobtained in phage form from the laboratory of Ronald Davis(Department ofBiochemistry, Stanford University) (13). Theplasmid portion (pSE937 with the inserted library DNA) wasexcised by using the automatic subcloning feature of theAYES system (14). Plasmid DNA from a total of 20 millionindependent colonies was used to transform strain SGL9 byelectroporation (15). Transformants were first selected foruracil prototrophy on medium lacking uracil (typical trans-formation efficiencies were 300 colony-forming units per ;Lg)and then for ergosterol prototrophy on medium supple-mented with methionine (0.01%) and hemin hydrochloride(12). Plasmid DNA was extracted from yeast by standardmethods (16) and transformed into Escherichia coli strainXL-1 Blue for larger-scale preparation and further analysis.E. coli was grown in LB medium supplemented with ampi-cillin (50 mg/l). DNA was isolated from bacteria by using thealkaline lysis method in conjunction with Qiagen (Chats-worth, CA) column purification.

Cydization Asay. Fifteen-milliliter cultures of strains 9aand SGL9 were grown to saturation (OD6No 2.5) in SDCmedium supplemented with ergosterol, Tween 80, and heminhydrochloride. Fifteen-milliliter cultures of strain SGL9-harboring plasmids pZS2, pZS4, or pZSl1 were grown tosaturation in medium supplemented with methionine and

Abbreviation: ORF, open reading frame.*To whom reprint requests should be addressed.

7370

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

2, 2

020

Proc. Natl. Acad. Sci. USA 91 (1994) 7371

hemin hydrochloride. The cells were collected by centrifu-gation, washed once with 1 ml of 100 mM potassium phos-phate buffer (pH 6.5), resuspended in three times the wetmass of phosphate buffer, and lysed by mixing in the pres-ence ofglass beads (425-600 pm, acid washed, Sigma), whichwere added to just below the liquid level. Fifty microliters ofeach lysate was then added to microcentrifuge tubes thatcontained 2,3-[3H]oxidosqualene (17) (50 nmol, 137 nCi; 1 Ci= 37 GBq) and Triton X-100 (0.5 A1). The reactions wereincubated at 250C for one-half day and then evaporated.Residues were extracted with ether (20 j1 twice), and theextracts were fractionated by silica gel TLC using hexane/ethyl acetate, 4:1 eluent. The TLC plate was sprayed withEN3HANCE (DuPont) and then visualized by autoradiogra-phy. The extent of conversion was determined by scintilla-tion counting of the silica gel containing oxidosqualene andlanosterol from each lane of the TLC plate.Sequencing and Sequence Analysis. The transposon-

mediated sequencing method (TN1000 kit, Gold Biotechnol-ogy, St. Louis) was used. Plasmid pZS3J was generated bycloning the complementing 3.4-kb Xho I fragment frompZS11 into the sequencing vector pMOB (18). A nested set ofpZS3J plasmids with transposon inserted every 200-300 ntwas then generated. Both strands of these constructs weresequenced by a combination of cycle sequencing (19) andautomated sequencing on an Applied Biosystems model 373ADNA sequencer. The sequence was assembled and analyzedby using MacVector software (version 3.04, IBI). Sequenceidentities among cyclase enzymes were determined withprogram GENALIGN (IntelliGenetics, release 5.4).Northern Hybridization. Poly(A)+ RNA from S. cerevisiae

(Clontech) was size-fractionated by electrophoresis on a0.8% agarose/2.2 M formaldehyde gel. The gel was treatedwith 0.05 M NaOH for 10 min, and the RNA was blotted ontoa Nytran membrane (Schleicher & Schuell) and then UVcross-linked using a Stratalinker model 1800 (Stratagene).The membrane was hybridized using 32P-labeled probesderived from the 486-bp EcoRI restriction fragment fromwithin the S. cerevisiae ERG7 gene and random nonadeox-ynucleotide primers (Stratagene) and then visualized byautoradiography.

Polyclonal Antibodies and Western Hybridization. Rabbitpolyclonal antisera directed against a predicted decapeptidesequence of the S. cerevisiae cyclase (DGGWGLHSVD,residues 140-149) were generated using an eight-branchedmultiple antigenic peptide and standard immunization proto-cols (20). IgG fractions of the antiserum were purified byaffinity chromatography on a Protein G column and stored at-20°C. Soluble proteins from glass-bead lysis of S. cerevisiaestrains 9a, SGL9, and SGL9 transformed with pZSll wereseparated by SDS/7.5% PAGE and immobilized on a nitro-cellulose membrane (Bio-Rad) by transfer electrophoresis.The membrane was blocked with 2% nonfat milk, probedwith affinity-purified rabbit anti-peptide IgG, and visualizedby using an aLkaline phosphatase-conjugated goat anti-rabbitIgG secondary antibody (Jackson ImmunoResearch) in con-junction with chromogenic phosphatase substrates (Bio-Rad).

RESULTS AND DISCUSSIONIsolatin of the S. cerevusiae ERG7 Gene by Complementa-

tion. The ERG7 gene from S. cerevisiae was isolated after thecomplementation approach used by Kelly et al. (7) to isolatethe ERG7 gene from C. albicans. The erg7 ura3-52 strainSGL9 was transformed with a S. cerevisiae genomic libraryin the URA3 shuttle vector pSE937 derived from AYES (13,14) by electroporation (15). Transformants were selected forgrowth in the presence of ergosterol but in the absence ofuracil. Uracil prototrophs (105) were then selected for growth

in the absence of both uracil and ergosterol. Three yeastcolonies were found to harbor plasmids (pZS2, pZS4, pZSll)that complemented the erg7 mutation upon retransformationof SGL9. Restriction analysis (data not shown) indicated thatpZSll carried a 3.4-kb insert in the pSE937 vector, whereaspZS2 and pZS4 carried a larger 4.8-kb insert that encom-passed the insert of pZSll.

Restoration of Oxldosqualene- anosterol Cyclase Activityby pZS2, pZS4, and pZSll. To determine whether and towhat extent pZS2, pZS4, and pZSll restored cyclase activityto strain SGL9, cell-free extracts from strain 9a (which bearsa wild-type ERG7 gene), strain SGL9, and strain SGL9transformants were assayed for their ability to convert 2,3-[3H]oxidosqualene to [3H]lanosterol (Fig. 2). No cyclaseactivity was observed in the cell-free extract from strainSGL9 (lane 2) or from SGL9 transformed with pSE937 alone(data not shown). Substantial cyclase activity was observedin extracts from 9a and from pZS2-, pZS4-, and pZSll-transformed SGL9 (lanes 1, 3-5). Quantification showed thatunder the conditions used the cell-free extract from strain 9aconverted 36% of racemic 2,3-[3H]oxidosqualene to [3H]-lanosterol. This result corresponds to a 72% conversion ofthe S-enantiomer of the substrate, for which the enzyme isspecific (21). Extracts from pZS2-, pZS4-, and pZSll-transformed SGL9 converted 31, 29, and 29%o of the racemicsubstrate to product, respectively. Thus, the inserts frompZS2, pZS4, and pZSll in the low-copy number, centromericplasmidpSE937 restore =80o ofwild-type cyclase activity tostrain SGL9. This result contrasts with the high-copy num-ber, YEp13-based complementing plasmids bearing theERG7 gene from C. albicans, which restore, at most, 60%6 ofwild-type S. cerevisiae cyclase activity (7) and suggests thatthe homologously expressed S. cerevisiae cyclase is presentat higher levels and/or is more stable or active than is the C.albicans cyclase when heterologously expressed in S. cere-visiae.DNA Sequence ofthe S. cerevisiaeERG7 Gene and Predicted

Amino Acid Sequence of the S. cerevisiae Oxidosqualene-Lanosterol Cyclase. We determined 2.9 kb ofDNA sequencefrom the 3.4-kb insert in pZSll (Fig. 3); this revealed apotential ORF of 2196 nt, including the stop codon. A

- *- Os

9^ 0 0 *

2 4

0N N

w -ac r CD

CM LI) O aUo Cf -

FIG. 2. Autoradiogram of TLC of products from cyclization of2,3-[3H]oxidosqualene in cell-free extracts of S. cerevisiae strains 9a(ERG7, lane 1), SGL9 (erg7, lane 2), and SGL9 transformed withcomplementing plasmids pZS2 (lane 3), pZS4 (lane 4), and pZSl1(lane 5). The mobilities of authentic standards ofoxidosqualene (OS)and lanosterol (L) are indicated with arrows.

Biochemistry: Shi et A

4ft do 40:40

Dow

nloa

ded

by g

uest

on

Mar

ch 1

2, 2

020

7372 Biochemistry: Shi et al.

1469113618122627131636140645149654158663167672176681185690194699110361081112611711216126113061351139614411486153115761621166617111756180118461891193619812026207121162161

Proc. Natl. Acad. Sci. USA 91 (1994)

CCGGTCAGTTACCTGTCATTGGTCAAATTTTCTTGCCCAATGACT

ERG7/S. c.

ERG7/C. a.

ERG7/S. c.

kRG7/C. a.

ERG7/S. c.

ERG7/C.a.

ERG7/S. c.

ERG7/C.a.

ERG7/S. c.

ERG7/C. a.

ERG7/S. c.

ERG7/C.a.

ERG7/S. c.

ERG7/C.a.

1

1

MTEFYSDTIGLPKTDPRLWRLRTDELGRESWEYLTPQQAANDPPS11 1111111 lIll I1111 I 11 I I

MYYSEEIGLPKTDISRWRLRSDALGRETWHYLSQSECESEPQS

46 TFTQWLLQDPKFPQPHPERNKHSPDFSAFDACHNGASFFKLLQEP11 III 11 11 MIii

44 TFVQWLLESPDFP-----SPPSSDIHTSGEAARKGADFLKLLQ-L

91 DSGIFPCQYKGPMFMTIGYVAVNYIAGIEIPEHERIELIRYIVNT

83 DNGIFPCQYKGPMFMTIGYVTANYYSKTEIPEPYRVEMIRYIVNT

136 AHPVDGGWGLHSVDKSTVFGTVLNYVILRLLGLPKDHPVCAKARS11111111111111111II11111 11111 11111 111

128 AHPVDGGWGLHSVDKSTCFGTTMNYVCLRLLGMEKDHPVLVKARK

181 TLLRLGGAIGSPHWGKIWLSALNLYKWEGVNPAPPETWLLPYSLP11 111111 1111111 111111111

173 TLHRLGGAIKNPHWGKAWLSILNLYEWEGVNPAPPELWRLPYWLP

226 MHPGRWWVHTRGVYIPVSYLSLVKFSCPMTPLLEELRNEIY---T11 111111 1 1 111 11111

218 IHPAKWWVHTRAIYLPLGYTSANRVQCELDPLLKEIRNEIYVPSQ

268 KPFDKINFSKNRNTVCGVDLYYPHSTTLNIANSLVVFYEKYLRNR

263 LPYESIKFGNQRNNVCGVDLYYPHTKILDFANS-ILSKWEAVRPK

ERG7/S.c. 313 FIYSLSKKKVYDLIKTELQNTDSLCIAPVNQAFCALVTLIEEGVD1111111 III 111111 11 11 11

ERG7/C.a. 307 WLLNWVNKKVYDLIVKEYQNTEYLCIAPVSFAFNMVVTCHYEGSE

ERG7/S. c.

ERG7/C.a.

ERG7/S. c.

ERG7/C.a.

358 SEAFQRLQYRFKDALFHGPQGMTIMGTNGVQTWDCAFAIQYFFVA11 11 I IIIIIIIIIIIIIIII 11 11 1ill

352 SENFKKLQNRMNDVLFHGPQGMTVMGTNGVQVWDAAFMVQYFFMT

403 GLAERPEFYNTIVSAYKFLCHAQFDTECVPGSYRDKRKGAWGFSTII I 11 11 11 11 11 11111 III

397 GLVDDPKYHDMIRKSYLFLVRSQFTENCVDGSFRDRRKGAWPFST

ERG7/S.c. 448 KTQGYTVADCTAEAIKAIIMVKNSPVFSEVHHMISSERLFEGIDVl 11111 111111 111111 I 11

ERG7/C.a. 442 KEQGYTVSDCTAEAMKAIIMVRNHASFADIRDEIKDENLFDAVEV

ERG7/S.c. 493 LLNLQNIGSFEYGSFATYEKIKAPLAMETLNPAEVFGNIMVEYPY11 11 I 11111 III 11111 1111111 11111111

ERG7/C.a. 487 LLQIQNVGEWEYGSFSTYEGIKAPLLLEKLNPAEVFNNIMVEYPY

GGTTGGGGGGAATCAATGAAGTCCAGTGAATTACATAGTTATGTGGATAGTGAAAAATCGCTAGTCGTTCAAACCGCATGGGCGCTAATT

FIG. 3. Nucleotide sequence of the S. cerevisiae ERG7 gene.Nucleotides of the open reading frame (ORF) are capitalized. Nu-cleotides of the 5'- and 3'-untranslated regions are in lowercaseletters. Numbering begins with the first nucleotide of the ORF.

consensus TATA box promoter sequence (22) and a consen-sus polyadenylylation signal (23, 24) were found before theinitiation codon and after the stop codon, respectively. TheORF is very similar in length to that of the C. albicans ERG7structural gene (2187 nt) (6), and these ORFs show 56%nucleotide sequence identity with few gaps. Based on theDNA sequence information and the ability of the pZS11insert to complement the erg7 mutation and restore nearwild-type levels of cyclase activity, we conclude that theisolated sequences bear the S. cerevisiae ERG7 gene.t

This gene encodes a predicted 83.4-kDa protein of 731amino acids, which is nearly identical to molecular massvalues predicted for the oxidosqualene-lanosterol cyclasefrom C. albicans (6) and the oxidosqualene-cycloartenolcyclase from A. thaliana (9), similar to the molecular mass

ERG7/S. c.

ERG7/C.a.

ERG7/S. c.

ERG7/C. a.

ERG7/S. c.

ERG7/C.a.

ERG7/S. c.

ERG7/C. a.

538 VECTDSSVLGLTYFHKY-FDYRKEEIRTRIRIAIEFIKKSQ-LPD

532 VECTDSSVLGLTYFAKYYPDYKPELIQKTISSAIQYILDSQDNID

581 GSWYGSWGICFTYAGMFALEALHTVGETYENSSTVRKGCDFLVSK11111 1111 III 11111111111 11 11 111111 11

577 GSWYGCWGICYTYASMFALEALHTVGLDYESSSAVKKGCDFLISK

626 QMKDGGWGESMKSSELHSYVDSEKSLWQTAWALIALLFAEYPNK11111111 1 1111 I I1 111HillI 11

622 QLPDGGWSESMKGCETHSYVNGENSLVVQSAWALIGLILGNYPDE

671

667l111 11 1111 1

EPIKRGIQFLMKRQLPTGEWKYEDIEGVFNHSCAIEYPSYRFLFP

ERG7/S.c. 716 IKALGMYSRAYETHTL11111

ERG7/C.a. 712 IKALGLYKNKYGDKVLV

FIG. 4. Alignment of predicted amino acid sequences of oxido-squalene-lanosterol cyclases from S. cerevisiae (S.c., upper se-quence) and C. albicans (C.a., lower sequence). Identities areindicated with vertical lines. Gaps introduced to maximize alignmentare indicated with hyphens.

values assigned to vertebrate oxidosqualene-lanosterol cy-clases (8), but inconsistent with the 26-kDa value assigned topurified S. cerevisiae cyclase (10). It is possible that the S.cerevisiae cyclase mRNA is spliced before expression-thesequence of the S. cerevisiae OR does have a consensus5-splice donor sequence for a yeast intron, although it occursuntypically far into the nucleotide sequence (positions 844-849) and is not accompanied by the strictly conserved yeastbranch-point sequence (26). With a probe derived from the S.cerevisiae ERG7 gene, a single transcript of 2.4 kb wasdetected by Northern hybridization of size-fractionated S.cerevisiae mRNA (data not shown), which is consistent withan unspliced message. It is also possible that the S. cerevisiaeenzyme is naturally processed to a smaller, catalyticallyactive form. However, the predicted N-terminal sequencedoes not resemble typical yeast signal sequences that could

tTbhis sequence is >99% identical to that recently reported by Coreyet al. (ref. 25; GenBank accession no. U04841), who isolated the S.cerevisiae ERG7 gene by hybridization methods.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

2, 2

020

Proc. Natl. Acad. Sci. USA 91 (1994) 7373

FIG. 5. Hypothetical model for involvement of electron-richaromatic side chains from tryptophan and tyrosine residues incyclization of oxidosqualene to the protosterol cation. Shown are ageneral acid catalyst (A-H) that initiates cyclization and phenol sidechains from tyrosine (OH) and an indole side chain from tryptophan(NH) that direct and accelerate cyclization through cation-Ir inter-actions.

target the enzyme to the lumen of the endoplasmic reticulumand/or to other organelles for processing (27), and no endo-plasmic reticulum retention signal is observed. at the Cterminus (28). The sequence does predict one reasonablyhydrophobic region 15-amino acid residues in length (resi-dues 651-665), which may be involved in membrane local-ization by the enzyme (29). Immunohybridization using rab-bit polyclonal IgG elicited against a predicted decapeptidesequence of the S. cerevisiae cyclase detects a specificprotein of Mr 80,000 in cell-free extracts of S. cerevisiae,which is not detected with preimmune antiserum (data notshown).The predicted amino acid sequence of the S. cerevisiae

cyclase exhibits 63% identity to that of the C. albicanscyclase (Fig. 4). The former also bears substantial identity tothe predicted amino acid sequences of the oxidosqualene-cycloartenol cyclase from A. thaliana (36%) (9) and thesqualene-hopene cyclase from Alicyclobacillus acidocaldar-ius (16%) (30). Observation of these identities supports theproposed divergent evolutionary relationship among cyclaseenzymes (31-33). Segments of the predicted amino acidsequences of the S. cerevisiae and C. albicans cyclases(residues 439-468 and 433-462, respectively) also show 50%oidentity to a peptide sequence from the active site of rat livercyclase that is labeled by the mechanism-based inactivator29-[3H] methylidene-2,3-oxidosqualene at each of the twoadjacent aspartyl (D) residues within the octapeptide se-quence VADDTAEA (34). The corresponding sequence inthe S. cerevisiae cyclase is predicted to be VADCTAEA(residues 454-461). Here, one of the aspartyl residues isconserved and the other is replaced with a cysteine residue,which also bears a potential side-chain nucleophile. As the S.cerevisiae cyclase is inhibited but not labeled by 29-[3H]methylidene-2,3-oxidosqualene (34), it appears thatrather subtle changes within this sequence and/or changesoutside of this sequence substantially affect susceptibility tothis inhibitor.The S. cerevisiae, C. albicans, A. thaliana, and A. aci-

docaldarius cyclases are predicted to contain unusually largeamounts oftryptophan and tyrosine, which bear electron-richaromatic side chains. With 2.3% tryptophan and 5.5% tyro-sine, the S. cerevisiae cyclase possesses each of these aminoacids at levels greater than those found in 95% ofS. cerevisiaeproteins (35). Strikingly, 16/17 (94%) of the tryptophanresidues and 34/40 (85%6) ofthe tyrosine residues found in theS. cerevisiae cyclase sequence are found at identical posi-tions within the C. albicans cyclase sequence. These findingsand consideration of the electrophilic nature of polyenecyclization/rearrangement reactions lead us to formulate anaromatic hypothesis for cyclase active-site structure. In thishypothesis, electron-rich indole and phenol side chains from

a subset of the conserved tryptophan and tyrosine residuesare essential features of cyclase active sites, where theydirect the folding of the substrate and, through cation-irinteractions, stabilize positively charged transition statesand/or high-energy intermediates during cyclization/rear-rangement. For example, they may constitute the "negativepoint charges" postulated by Johnson etaL (36-39) to controlcyclization regio- and stereochemistry through stabilizationof specific cationic transition states and high-energy inter-mediates (Fig. 5). Cation-ninteractions are common featuresof receptor-ligand and enzyme-substrate complexes (40-43)and have been found to stabilize both ground and transitionstates in biomimetic catalyst systems (44, 45).

We are most grateful to Dr. John McCusker and Mr. Helge Zielerfor advice and expert technical assistance and to Mr. David Eschel-bacher for preparing 2,3-[3H]oxidosqualene. This work was sup-ported by Stanford University, a starter grant from the NationalScience Foundation (CHE-9018241), A Young Investigator Awardfrom the Arnold and Mabel Beckman Foundation, a Camille andHenry Dreyfus Foundation New Faculty Award, a Shell FoundationFaculty Career Initiation Award, an Eli Lilly Graduate ResearchFellowship to Z.S., and a National Science Foundation PredoctoralFellowship to C.J.B.

1. Abe, I., Rohmer, M. & Prestwich, G. D. (1993) Chem. Rev. 93,2189-2206.

2. Robinson, R. (1934) J. Soc. Chem. Ind. 53, 1062-1063.3. Woodward, R. B. & Bloch, K. (1953) J. Am. Chem. Soc. 75,

2023-2024.4. Eschenmoser, A., Ruzicka, L., Jeger, 0. & Arigoni, D. (1955)

Helv. Chim. Acta 38, 1891-1904.5. Stork, G. & Burgstahler, A. W. (1955) J. Am. Chem. Soc. 77,

5068-5077.6. Buntel, C. J. & Griffin, J. H. (1992) J. Am. Chem. Soc. 114,

9711-9713.7. Kelly, R., Miller, S. M., Lai, M. H. & Kirsch, D. R. (1990)

Gene 87, 177-183.8. Abe, I., Bai, M., Xiao, X.-y. & Prestwich, G. D. (1992)

Biochem. Biophys. Res. Commun. 187, 32-38.9. Corey, E. J., Matsuda, S. P. & Bartel, B. (1993) Proc. Natl.

Acad. Sci. USA 90, 11628-11632.10. Corey, E. J. & Matsuda, S. P. T. (1991) J. Am. Chem. Soc.

113, 8172-8174.11. Corey, E. J., Virgil, S. C. & Sarshar, S. (1991) J. Am. Chem.

Soc. 113, 4025-4026.12. Gollub, E. G., Liu, K.-P., Dayan, J., Adlersberg, M. & Spmn-

son, D. B. (1977) J. Biol. Chem. 252, 2846-2854.13. Ramer, S. W., Elledge, S. J. & Davis, R. W. (1992) Proc. Nat!.

Acad. Sci. USA 89, 11589-11593.14. Elledge, S. J. Mu , J. T., Ramer, S. W., Spottswood, M.

& Davis, R. W. (1991) Proc. Natl. Acad. Sci. USA 88, 1731-1735.

15. Becker, D. M. & Guarente, L. (1991) Methods Enzymol. 194,182-187.

16. Strathern, J. N. & Higgins, D. R. (1991) Methods Enzymol.194, 319-329.

17. Nadeau, R. G. & Hanzlik, R. P. (1969) Methods Enzyumol. 15,346-351.

18. Strathmann, M., Hamilton, B. A., Mayeda, C. A., Simon,M. I., Meyerowitz, E. M. & Palazzolo, M. J. (1991) Proc.Nat!. Acad. Sci. USA 88, 1247-1250.

19. Murray, V. (1989) Nucleic Acids Res. 17, 8889.20. Tam, J. P. (1988) Proc. Natl. Acad. Sci. USA 85, 5409-5413.21. Barton, D. H. R., Jarman, T. R., Watson, K. C., Widdowson,

D. A., Boar, R. B. & Damps, K. (1975) J. Chem. Soc. PerkinTrans. 1, 1134-1138.

22. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. 50,349-383.

23. Proudfoot, N. J. & Brownlee, G. G. (1976) Nature (London)263, 211-214.

24. Fitzgerald, M. & Shenk, T. (1981) Cell 24, 251-260.25. Corey, E. J., Matsuda, S. P. T. & Bartel, B. (1994) Proc. Nat!.

Acad. Sci. USA 91, 2211-2215.26. Woolford, J. L., Jr. (1989) Yeast 5, 439-457.

Biochemistry: Shi et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

2, 2

020

7374 Biochemistry: Shi et al.

27. von Hejne, G. (1985) J. Mol. Biol. 184, 99-105.28. Peiham, H. R. B., Hardwick, K. G. & Lewis, M. J. (1988)

EMBO J. 7, 1757-1762.29. Hoshino, T., Williams, H. J., Chung, Y. & Scott, A. I. (1991)

Tetrahedron 47, 5925-5932.30. Ochs, D., Kaletta, C., Entian, K.-D., Beck-Sickinger, A. &

Poralla, K. (1992) J. Bacteriol. 174, 298-302.31. Rohmer, M., Bouvier, P. & Ourisson, G. (1979) Proc. Nati.

Acad. Sci. USA 76, 847-851.32. Ourisson, G., Rohmer, M. & Poralla, K. (1987) Annu. Rev.

Microbiol. 41, 301-333.33. Ourisson, G. (1989) Pure Appl. Chem. 61, 345-348.34. Abe, I. & Prestwich, G. D. (1994) J. Biol. Chem. 269, 802-804.35. Karlin, S., Blaisdell, B. E. & Bucher, P. (1992) Protein Eng. 5,

729-738.36. Johnson, W. S., Telfer, S. J., Cheng, S. & Schubert, U. (1987)

J. Am. Chem. Soc. 109, 2517-2518.

Proc. Nadl. Acad. Sci. USA 91 (1994)

37. Johnson, W. S., Lindell, S. D. & Steele, J. (1987) J. Am. Chem.Soc. 109, 5852-5853.

38. Johnson, W. S. (1991) Tetrahedron 47, xi-1.39. Johnson, W. S., Buchanan, R. A., Bartlett, W. R., Tham,

F. S. & Kullnig, R. K. (1993) J. Am. Chem. Soc. 115, 504-515.40. Burley, S. K. & Petsko, G. A. (1986) FEES Lett. 203, 139-143.41. Burley, S. K. & Petsko, G. A. (1988) Adv. Protein Chem. 39,

125-189.42. Davies, D. R. & Metzger, H. (1983) Annu. Rev. Immunol. 1,

87-117.43. Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman,

A., Toker, L. & Silman, I. (1991) Science 253, 872-879.44. Dougherty, D. A. & Stauffer, D. A. (1990) Science 250, 1558-

1560.45. McCurdy, A., Jimenez, L., Stauffer, D. A. & Dougherty, D. A.

(1992) J. Am. Chem. Soc. 114, 10314-10321.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

2, 2

020