Proc. Natl. Acad. Sci. USA Vol. 92, pp. 9274-9278, September 1995 Biochemistry

Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA ( biosynthesis/cDNA cloning/active site/QW motif) IKURO ABE AND GLENN D. PRESTWICH* Department of Chemistry, State University of New York, Stony Brook, NY 11794-3400 Communicated by William J. Lennarz, State University of New York, Stony Brook NY, July 3, 1995 (received for review May 10, 1995)

ABSTRACT A cDNA encoding rat oxidosqualene lano- 5.4.99.-) have been cloned from Alicyclobacillus acidocaldarius -cyclase [ synthase; (S)-2,3-epoxysqualene (21) and Zymomonas mobilis (22). The predicted molecular mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was masses of OSCs ranged from 80 to 90 kDa and the deduced cloned and sequenced by a combination of PCR amplification, amino acid sequences showed that the bacterial, fungal, and using primers based on internal amino acid sequence of the plant share a very ancient ancestry. A highly con- purified , and cDNA library screening by oligonucle- served repetitive (3-strand turn motif rich in aromatic amino otide hybridization. An open reading frame of2199 bp encodes acids (the QW motif) occurs in all OSCs and SCs, and this a Mr 83,321 protein with 733 amino acids. The deduced amino likely serves a structural or catalytic role in the cyclization acid sequence of the rat enzyme showed significant homology reaction (23, 24). We previously reported mapping of the to the known oxidosqualene cyclases (OSCs) from yeast and active site of rat liver OSC using 29-[3H]methylidene-2,3- plant (39-44% identity) and still retained 17-26% identity to oxidosqualene ([3H]29-MOS), a mechanism-based irreversible two bacterial cyclases (EC 5.4.99.-). Like other inactivator specific for vertebrate OSCs (8, 25-27). We de- cyclases, the rat enzyme is rich in aromatic amino acids and scribe here the cDNA cloning and characterization of rat liver contains five so-called QW motifs, highly conserved regions OSC, and we report the expression of a functional rat OSC in with a repetitive 13-strand turn motif. The binding site se- the sterol auxotrophic SGL9 yeast strain.t quence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the ver- tebrate cyclase, is well-conserved in all known OSCs. The EXPERIMENTAL PROCEDURES hydropathy plot revealed a rather hydrophilic N-terminal Amino Acid Analysis and Protein Sequencing of Rat OSC. region and the absence of a hydrophobic signal peptide. Purified rat liver OSC was labeled with [3H]29-MOS and was Unexpectedly, this microsomal membrane-associated enzyme digested with CNBr or with endoproteinase Lys-C and se- showed no clearly delineated transmembrane domain. A full- quenced by Edman degradation as described (26). The fol- length cDNA was constructed and subcloned into a pYEUra3 lowing partial sequences were obtained: CNBr 8-kDa frag- plasmid, selected in Escherichia coli cells, and used to trans- ment, VRYLRSVQLPDGGWGLHHEDKSTVFG (aa 129- form the OSC-deficient uracil-auxotrophic SGL9 strain of 154); Lys-C 50-kDa fragment, NNVCPDDMY (aa 279-287); Saccharomyces cerevisiae. The recombinant rat OSC expressed CNBr 6-kDa fragment, HKGGFPFSTLDDGWIVADDTAE- was efficiently labeled by the mechanism-based inhibitor ALKAVLLLQE (aa 439-470). [3H]29-MOS. Library Construction and Screening. Male Sprague- Dawley rats were fed with cholestyramine and Fluvastatin Oxidosqualene lanosterol-cyclase (OSC) [; (hydroxymethylglutaryl-CoA reductase inhibitor) for 1 week (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), to induce 15-fold higher OSC activity relative to control rats EC 5.4.99.7] catalyzes the conversion of (3S)-2,3-oxidosqua- (28). From the induced liver, total RNA was obtained by the lene to lanosterol, forming a total of six new carbon-carbon acid guanidium thiocyanate/phenol/chloroform extraction bonds in a single reaction (1). The regulation of OSC levels in method (29). A cDNA library was then constructed from 5 ,g vivo has clinical importance and has been a potential target for of poly(A)+ mRNA with the ZAP cDNA synthesis kit with the design of hypercholesteremic drugs (2). The formation of Lambda Uni-ZAP XR vector and the Gigapack II packaging lanosterol is initiated in the chair-boat-chair-like conformation extract (Stratagene). The titer of the resulting library was 7.5 of oxidosqualene, and the proton-initiated cyclization is pos- X 105 plaque-forming units/Al after one amplification. tulated to proceed through a series of rigidly held carbocat- For screening, a PCR amplified 500-bp cDNA fragment ionic intermediates (1). The intermediate C-20 protosterol obtained as described below was labeled with [a-32P]dATP. cation then undergoes backbone rearrangement to yield the Recombinant plaques (41 x 106) were transferred to nylon lanosterol skeleton (Fig. 1) (3-6). Oxidosqualene is also the membranes (Hybond-N; Amersham) and hybridized with the precursor for a variety of other polycyclic , and the radiolabeled probe in 5 x SSPE (0.75 M NaCl/0.05 M relationship between enzyme structure and cyclization mech- NaH2PO4/5 mM EDTA, pH 7.4)/Sx Denhardt's reagent/ anism has been extensively studied (1). 50% formamide/0.1% SDS/100 ,tg of salmon testes DNA per Several OSCs have been purified to homogeneity from ml at 42°C for 24 hr. Membranes were washed three times with vertebrate (7-9), plant (10-13), and yeast sources (14). Re- 0.1 x SSC (15 mM NaCl/1.5 mM sodium citrate, pH 7.0)/0.1% cently, two OSC enzymes have been cloned and sequenced SDS at 55°C for 20 min. Two positive phage clones were from the fungi Saccharomyces cerevisiae (15, 16) and Candida purified and subcloned into Bluescript II SK- phagemids. One albicans (17-19). An oxidosqualene cycloartenol-cyclase was an cDNA insertion. cloned and sequenced from the plantArabidopsis thaliana (20). of the positive clones contained -2-kbp In addition, two bacterial squalene hopene-cyclases (SCs) (EC Abbreviations: SC, squalene cyclase; OSC, oxidosqualene cyclase; 29-MOS, 29-methylidene-2,3-oxidosqualene. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U31352). 9274 Downloaded by guest on October 2, 2021 Biochemistry: Abe and Prestwich Proc. Natl. Acad. Sci. USA 92 (1995) 9275

Enz-B:' , Enz-AHA H HO -3. -'- HO Protostrol catiH

(3S)2,3-Oxidosqualene L Protosterol cation Lanosterol FIG. 1. Formation of lanosterol from (3S)-2,3-oxidosqualene by OSC. Both strands of cDNA were completely sequenced by the downstream of the yeast GAL] promoter. After transforma- dideoxynucleotide chain-termination method using Sequenase tion of Escherichia coli DH5a cell and selection on LB/ version 2.0 (United States Biochemical) (30). ampicillin plates, plasmids were recovered and checked for the PCR Screening of cDNA Library. Degenerate oligonucleo- insertion. The SGL9 strain was then transformed with the tides 5'-TG(T/C)CCNGA(T/C)GA(T/C)ATGTA-3' (sense) recovered plasmid by the LiAc method (34), and transformants coding CPDDMY of the Lys-C 50-kDa fragment (aa 282-287) were selected on synthetic complete (SDC) medium without and 5'-TCNGCNAC(G/A/T)ATCCANCC-3' (antisense) uracil and ergosterol. coding GWIVAD of the CNBr 6-kDa fragment (aa 451-456) Enzyme Assay and Affinity Labeling. The Ural Erg' yeast were used for amplification of a partial sequence of the cDNA transformant cell was first grown in 50 ml of SDC medium (524 bp). After phage particles were disrupted (70°C for 5 min containing 2% glucose. When cells grew to OD600 = 0.5, the and then 4°C), 30 cycles of PCR were carried out as follows: medium was replaced with the SDC medium containing 2% 1 min at 94°C, 2 min at 45°C, and 3 min at 72°C. The galactose and incubated for another 3 hr to induce expression gel-purified PCR products were ligated into pGEM-T plasmid of the OSC gene under the GALl promoter. The cell pellet (Promega) and sequenced. (100 mg) obtained after centrifugation was washed once with To obtain a full-length sequence, PCR amplification of the 100 mM Tris-HCl (pH 7.4), resuspended in 200 ,ul of 100 mM 5' end of the cDNA from the library was first performed using Tris'HCl (pH 7.4) containing 1 mM dithiothreitol, and then three primer sets of gene-specific internal antisense primers broken by Vortex mixing with glass beads (eight times for 45 and nonspecific T3 primer coding part of the A ZAP vector arm s). The cell-free extracts (100 ,ul) were incubated with (3S)2,3- (primer ratio, 10:1). Primer sets amplified 1000-, 570-, and [14C]oxidosqualene (50 ,uM; 5.5 mCi/mmol; 1 Ci = 37 GBq) 200-bp fragments, each with the same 5' end sequence and or [3H](3S)29-MOS (1 ,uM; 1.8 Ci/mmol) as described at 30°C each missing several 5' nucleotides required for an expected for 17 hr (8). For control experiments, the SGL9 strain alone full-length cDNA. and the SGL9 strain transformed with pYEUra3 plasmid To obtain the full 5' sequence, the 5' inverse PCR method lacking the rat OSC insert were used. was used (31, 32). Poly(A)+ mRNA (10 ,ug) was first reverse- transcribed by avian myeloblastosis virus reverse transcriptase (200 units), using 50 pmol of gene-specific antisense primer RESULTS AND DISCUSSION 5'-ATGGCCACTGCACCACCTT-3' (nt 551-569) instead of Rat OSC cDNA was cloned and sequenced from a rat liver the oligo(dT) primer. The reaction was run at 50°C for 1 hr to cDNA library by a combination of PCR amplification based on minimize the secondary structure of the RNA. After second- partial amino acid sequence of the purified enzyme followed strand DNA synthesis, the cDNA was circularized by T4 DNA by cDNA library screening with labeled oligonucleotides for ligase (30 units) at 15°C for 50 hr. PCR was then performed hybridization. The 2874-bp cDNA contained a 41-bp 5' non- by using primers 5'-AAGTCCATCTCTGCCGAC-3' (anti- coding region, a 2199-bp open reading frame encoding a Mr sense, nt 95-112) and 5'-CGTGTCACATAGCACAC-3' 83,321 protein of 733 amino acids, and 634 bp of 3' noncoding (sense, nt 335-351) to amplify a 388-bp DNA fragment region (Fig. 2). A polyadenylylation signal (AATAAA) pre- containing the 5' end sequence and an additional 41 bp of 5' cedes the poly(A) sequence by 27 bp, and an in-frame stop noncoding sequence. codon is located 36 bp upstream of the start codon. The Northern Blot Analysis. Poly(A)+ mRNA from rat liver was predicted molecular mass of 83 kDa is consistent with the size-fractionated by electrophoresis on a 0.8% agarose gel molecular size of the purified protein (78 kDa) estimated by containing 2.2 M formaldehyde. After the gel was treated with SDS/PAGE (8). In addition, the sequence (underlined) sur- 0.05 M NaOH, the denatured RNA was transferred to nylon rounding the start codon (boldface), GCT.TCATGA showed membrane and hybridized with a 32P-labeled cDNA probe good homology to the Kozak consensus sequence, iCC(Gi/ (using the 5' end 200-bp PCR product) and autoradiographed A)CCATG(G/A), for the 5' noncoding sequence ofvertebrate as described above. mRNA (35). Finally, on Northern blot analysis of rat liver Expression of Recombinant Rat OSC in Yeast. A yeast/E. poly(A)+ mRNA, a single transcript of -3 kb was detected coli shuttle vector pYEUra3 (Clontech) was used for func- (data not shown). tional expression of the rat OSC cDNA in the yeast OSC- The deduced amino acid sequence showed significant sim- deficient mutant strain SGL9 (erg7 ura3-52 hem3-6 gal2) that ilarity to the yeast and plant OSCs: 40.2% identity (295/733) was obtained from J. H. Griffin (16). The strain SGL9 is a with S. cerevisiae OSC, 39.0% identity (286/733) with C. segregant of a cross between OSC-deficient mutant GL7 strain albicans OSC, and 44.2% identity (324/733) with A. thaliana (erg7-s hem3-6 ga12) (33) and strain 9a (ura3-52). A full-length OSC (). It is interesting that rat lano- cDNA (2833 bp) was obtained by ligating the N-terminal PCR sterol synthase showed highest similarity to plant cycloartenol fragment (nt 1-1310) and the C-terminal fragment (nt 1311- synthase. The postulated cyclization mechanism of oxidosqua- 2833) excised from the above described 2-kbp Bluescript lene to cycloartenol is essentially the same as that for lanosterol insertion byNde I/Xho I digestion. Here the PCR primers used formation, except for the final 9f3,19-cyclopropane ring closure are 5'-AAGGATCC ATG ACC GAG GGC ACG TGT CTG instead of C-9 protein elimination (1). Only a slight modifi- C-3' (sense) (the BamHI site is underlined) and 5'-G GAA cation of the active site of the enzyme could determine the ACC ACC CTT GTG CAT ATG GCG-3' (antisense) (the Nde product specificity. Rat OSC also showed substantial identity I site is underlined). The amplified DNA was digested with to two prokaryotic SCs that directly cyclize squalene into the BamHI/Nde I prior to the ligation reaction. The cDNA was pentacyclic hopene: 26.3% identity (193/733) with cloned into the BamHI/Xho I site of the plasmid pYEUra3 Z. mobilis SC and 16.6% identity (122/733) with thermoaci- Downloaded by guest on October 2, 2021 9276 Biochemistry: Abe and Prestwich Proc. Natl. Acad. Sci. USA 92 (1995)

-41 GGTAAAGGGCTGGCCGGCCGGTGGTCCAGAGCTGTGCTGTC 1201 GGTGCACACCGAAGACCTGAGOT'TrOCCCTGCCTGCAGAAGGCTCACGAGTTCCTGCGG G A H R R P E F L P C L Q K A H E F L ATGACCGAGGGCACGTGTCTGCGGCGTCGTGGGGGACCCTATAAAACTGAGCCCGCCACC R M T E G T C LR R R GG P Y K T E P A T 1261 CTTTCCCAGGTCCCAGACAACAATCCTACTACCAGAAGTATTATCGCCATATGCACAAG L S Q V P D N N P D Y Q K Y Y H K 61 GATCTCACCCGCTGGCGGCTCCATAATGAGTTGGGTCGGCAGAGATGGACTTATTATCAA R H M D L T R W R L H N E L G RQ R W T YY Q 1321 GGTTTTCCCCTTCAGCACACTGGACTGTGGCTGGATCGTTGCTGACTGCACGGCCGAG 121 G G F P F S T L D C G W I V A D C T ARE A E E D P G RE Q T G L E A H S L G L D CNBr-6 kDa fragment * 29-NOB Binding alto 1381 GCTTTGAAGGCTGTGCTGCTCCTGCAGGAGCGGTGTCCCTCAATCACCGAGCATGTCCCC 181 ACAACAAGTTATTTCAAGAACTTACCTAAAGCTCAAACAGCCCATGAGGGGGCCCTGAAC A L K A V L LL O R RC P S I T E H V P T T S Y F KN L P K A Q T A H E G A L N 241 1441 CGAGAGCGACTCTACGATGCTGTGGCTGTGTTGTTGAGCATGAGGAATTCCGATGGAGGG GGAGTAACCTTTTATGCCAAGCTGCAGOCTGAGGATGGACACTGGGCTGGTGATTATGGT R E R L Y D A V A V G V T FY A K L Q A E D G H W A G D Y G L L S M R N S D G G 1501 301 ------...... L I I F AT Y E T K G G G P L F L L P G L L I T C H I A H I P L R Y L LE L L N P S E

361 1561.~~~~~~~ __.____.------CCGGC¶IGGATACAGAGAGGAAATGGTACGGTACTTGCGCTCAGOTCAGCTrCCCAATGGC V F G D I M I D Y T Y V P A G Y R E E MV R Y L R S V O L P N G E C T S A V M Q CNBr-U kDa fragmnt 1621 GCCCTGAGGCACTTCCGCGAGTACTTCCCAGACCACAGGGCTACAGAGATCAGGGAGACC 421 GGCTOOGGCTTGCACATTGAGGACAAGTCCACGOGTTTGGACACTGCCCTGAGCTATGTG A L R H FR E Y F P D H R A T E I R E T G W G L H I E D K S T V F G T A L S Y V 1681 481 TCTCTCAGAATCCTGGGTATTOGACCTGATGATCCTGACCTGTTGCGTGCTCGGAACATT L N Q G L D F C R K K Q R A D G S W E G S L R I L G I G P DD P D L V R A R I N 1741 TCCTOOOGGTTGuTTCACCTATGGCACCT3GGCTTGGAAGCATTCGCTTGCATO 541 CTTCACAAAAAAGGTGGTGCAGTGGCCATCCCTTCCTGOGGGAAGTTCTGGCTGGCTGTC S W G V C F T Y G T W F G L E A F A C M L H K K G G A V A I P S W G K F W L A V 1801 GGACATATCTACCAAAATAGGACTGCTTIGTGCAGAAGTAGCTCAGGCCTGCCACTTCCTC 601 CTGAATGTTTACAGCTGGGAAGGAATCAATACCCTCTTCCCTGAGATGTGGCTGCTTCCT G H I Y Q N R T A C A E V A Q A C H F L L N V Y S W E G I NT L F P E M W L L P 1861 CTGTCGCGGCAGATGGCGGAT,GGGGCTGGGGGGAGGACTTTGAGTCCTGTGAGCAGCGG 661 GAATGGTTTCCTGCACATCCCTCCACTCTGTGGTGTCACTGCCGGCAGGTCTATCTGCCC L S R Q M A D G G W G E D F E S C E Q R E W F P A H P S T L W C H C R Q V Y L P 1921 CGGTACGTGCAGAGTGCCGGGTCCCAGGTCCATAGTACGTGCTGGGCCCTGCTGGGTTT 721 R Y V Q S A G S Q V H S T C W A L L G L M S Y C Y AT R L S A S E D P L V Q S L 1981 781 CGCCAGGAACTCTATGTGGAGGATTATGCCAGCATCGATTIGGCCAGCACAGAAGAACAAC M A V R H P D I S A Q E R G I R C L L G R E L Y Q V E D Y A S I D W P A Q K N 2041 AAACAGCTCCCCAACGGAGACTGGCCTCAGGAGAACATCTCTGGGGTCTTCAACAAGTCC K Q LP N G D W P Q E N I S G V F N K S 841 GTGTGCCCCGATGACATGTACACGCCACACAGCTGGCTGCTGCACGTOGTATATGGACTC V C P D M Y T P H S W L L H V V Y G L 2101 TGTGCCATCAGCTACACAAATTACAGAAACATCTTCCCCATCTGGGCCCTCGGCCGCTTC Lys-C-50 kDa fragmnt CA I S Y T N Y R N I F P I W A L G R F 901 CTCAACCTGTATGAACGTTTCCACAGTACCAGCCTGCGGAAGTGGGCCATCCAGTTrCTG L N L Y E R F H S T S L R K W A I Q L L 2161 S S L YP D N T L A G H I 961 TATGAACATGTCGCAGCTGATGATCGGTTCACGAAATGCATCAGCATTGGCCCGATCTCA Y E H V A AD D R F T K C I S I G P I S 2221 GGCACCTGGCCAGCGTCAGCACTGTACAGGCCCAGGCAGCCCGGTTTCTCAGCTGGGTGA 2281 TAAGCGTCCTCCTAGCCCACTTTGGCATGGTTCTCTATTTCTGTAGACATGAGGCTGGG 1021 AAAACTGTCAACATGCTTATTCGTTGGTCAGTGGACGGACCATCCTCCCCTGCCTTCCAG 2341 ATAGGGTCAGAGTCGGTGTCTAGACACTGGGCAGCATGCGCTCGGTTCTGACGCTAGCTT K T V N M L I R W S V D G P S S P A F Q 2401 CAGTGAGGAAGGGAAGAAGTTGAGCTTGAGATCCAGAGAGGCACAGTGTACTCCGAGGCT 2461 CCTGGGTTCCTCCTTGAGGGAACAAGACTGTAGAGCTTTCATGATCAGTGACTTCCCTG 1081 GAGCACGTCTCGAGGATCAAAGATTATCTTTGGCTGGGCCTTGACGGCATGAAAATGCAG 2521 TGAGGGAGCAGCTGGCTTCTTCACTTCCTCCCTTGTTGACTGTGGAGTGGGCCCCGTGGT E H V S R I K DY L W L G L D G M K M Q 2581 GCAGGCATGCTCAGAGAAAGGCTCAATGGAGGAGGCAGGCCCCTTGCTCATTGTGTCCTG 2641 AGCAGTGTCTGTTGTCCCAGGCACTTACCCATGACTGC TCACTGCACGAAATACCAGTT 1141 2701 TTCCAGAAAGGAAGTACAAACTCGCTCTGTACTAAAAATGCTACTAAAAGTCGTTTGAAT G T N G S Q T W D T S F A V Q A LL E A 2761 GATCACCAACTCCCTTA&TAAACAGACCCCTGATGGCCCAGTAGTAAACAAAAAAA 2821 AAAAAAAAAAAAA 2833

FIG. 2. Nucleotide and predicted amino acid sequence of rat liver OSC. A 2199-bp open reading frame encoding 733 aa is shown with the single-letter code used for the translated amino acids. Nucleotide numbering begins with the ATG start codon. Amino acid sequences of peptides obtained from CNBr and Lys-C cleavages are underlined.

dophilicA. acidocaldarius SC. In general, the sequence is more of the developing cationic centers on the cyclizing substrate. highly conserved in the C-terminal region than in the N The electron density required for stabilization could arise from terminus. Except for these cyclases, no other significant se- anionic or from aromatic residues. The higher conservation of quence similarity was obtained from the GenBank/EMBL aromatic residues relative to anionic residues is noteworthy in data bases. view of the suggestion that cation-7r interactions may stabilize From sequence comparisons of eukaryotic OSCs and bac- cyclization intermediates (16, 23). Interestingly, an anionic terial SCs, we have previously reported the existence of the residue, D-456, is implicated in stabilization of the C-20 cation QW motif (23), a highly conserved repetitive motif rich in after tetracyclization but prior to hydride methyl migrations aromatic amino acids: [(K/R)(G/A)XX(F/Y/W)(L/I/ (26, 27). Furthermore, there are six conserved Cys (C-282, V)XXXQXXXGXW]. There are six repeats of the QW motif C-457, C-534, C-585, C-617, C-701, one at the 29-MOS binding in rat OSC, four in the C-terminal one-third and two in the site), and four conserved His (H-145, H-226, H-233, H-290) N-terminal one-third of the protein (Fig. 3). The motif was residues. The presence of an essential cysteinyl group in the well-conserved in both eukaryotic OSCs and prokaryotic SCs. active site of the enzyme has been previously suggested, since According to the PEPPLOT program (36), a typical QW motif the OSC activity can be efficiently inhibited by SH reagents contains part of a (3-strand at the N terminus and a turn at the such as p-chloromercuribenzenesulfonic acid and N- C terminus. We (37) and others (16) have postulated that the ethylmaleimide (13,42,43). In contrast, diethyl pyrocarbonate, aromatic amino acids of the QW motif might play a structural a histidyl-selective reagent, does not inhibit OSC activity (42, or functional role in catalysis by stabilizing the carbocationic 43). Finally, a disproportionately higher number of Gly (29 of intermediates through cation-I interactions. 59 residues} and Pro (11 of 38 residues) are also conserved, Of 733 amino acids of rat OSC, 175 residues (24%) are suggesting important conserved elements of secondary struc- completely conserved in all four known OSCs (rat, yeast, ture. Candida, plant). Overall, rat OSC contains a disproportion- According to Kyte-Doolittle hydropathy plot analysis (44), ately higher number of aromatic amino acid residues that are rat OSC is a moderately hydrophilic protein (Fig. 4). It has a completely conserved (16): Phe (13 of 28 residues), Trp (13 of hydrophilic region at the N terminus (D-5-E-53) as found in 24 residues), and Tyr (13 of 35 residues). The negatively the yeast and plant cyclases. Furthermore, no signal peptide charged Asp and Glu residues in rat OSC are less highly sequence was observed at the N terminus. Surprisingly, there conserved; Asp (6 of 36, 1 at the 29-MOS binding site) and Glu were no significantly hydrophobic regions that may serve as (10 of 43). As proposed by Johnson and colleagues (38-41), possible membrane-spanning regions. Indeed, the EMBL neu- negative point charges at the active site of the enzyme could ral network program (45, 46) predicted the absence of helical control the course of the cyclization reaction by stabilization transmembrane domains at the 95% confidence level. The Downloaded by guest on October 2, 2021 Biochemistry: Abe and Prestwich Proc. Natl. Acad. Sci. USA 92 (1995) 9277

QW-1 Rat 673 RG I R C L L G K Q L P NOD W A Rat 675 RG I DL L K N R SQ E W Yeast GEE Hydrophilicity window size = 15 Scale = Kyte-Doolittle Candida 671 RG I Q FL M KR Q L PT GE W Plant 702 Rh A R Y L I N A OMEN GD F 4.00- Bacteria-a 572 RG V Q Y LV E T Q R P D G G W Bacteria-z 595 KG I NW LA Q NQDE E G LW

QW-2 Rat 615 QAC HFLLSRQMADGGW Yeast 617 KGc c D F LV S K Q M K D G W 613 KG C D FL I S KQ L P D G G W Candida 11 200 0 500 600 700 Plant 640 RA C E FL L S KQ Q PS G G W 410 Bacteria-a 514 RA L DWV EQ H Q N P DGGW Bacteria-z 536 KR VA W L KT I QN EDO G W B Yeast Hydrophilicity window size = 15 Scale = Kyte-Doolittle QW-3 Rat 563 QGLDFCRKKQRADOSW Yeast 568 IAI EF IKKSQLPDGSW (3 Candida 563 SAIQYILDSQDNIDGSW 0. Plant 591 RAVKFIESIQAADGSW 2 0.00 Bacteria-a 466 Rh V E Y L K RE QK P DO SW -2.00 Bacteria-z 488 AAVDYLLKEQE E DGSW I -4.00] QW-4 Rat 486 DAVAVLLSMRNSDGGF )0 200 300 400 50o 600 760 Yeast 488 EGIDVLLNLQN-IaSF Candida 482 DAVEVLLQ IQN- VGEW Plant 514 EAVNVIISLONADGGL C Plant Bacteria-a 398 KGFRWIVGMQS SNGGW Hydrophi city window size = 15 Scale = Kyte-Doolittle Bacteria-z 420 RAMEWT I GMQS DN0GW 4.00] QW-5 Bacteria-a 332 KAGEWLLDRQITVPGDW Bacteria-z 350 SAL SWLK PQQ I LDVK¢DW -2.00- QW-6 Rat 127 EMVRYLRSVOLPNGGW -4.00- Yeast 127 ELIRYIVNTAHPVDGGW Candida 119 EM I RY IVNTAH PVDGGW 1oo 200 300 400 500 600 700 Plant 149 EMRRYLYNHQNEDOGW Amino acid residue QW-7 Rat 80 NOVTFYAKLQAEDGHW Yeast 79 NGA SF FKLLQE P DSG IF FIG. 4. Hydropathy plots for rat OSC (A), yeast S. cerevisiae OSC Candida 72 KRAD FL K LL QLD N 0I F (B), and plant A. thaliana cycloartenol synthase OSC. (C) Deduced Plant 99 RG L D F Y ST IQAH DGH W amino acid sequence was analyzed by MACVECTOR 4.1 sequence Bacteria-a 17 RA V E Y L L SC QK D E 0 Y W analysis software (Kodak). The QW motifs and the 29-MOS binding Bacteria-z 24 IATRALLEKQQQDHNW site (DCTAEA) are indicated. Plant and yeast OSCs are not labeled by [3H]29-MOS (8). FIG. 3. Summary of the highly conserved repetitive QW motifs [(K/R)(G/A)XX(F/Y/W)(L/I/V)XXXQXXXGXW] in OSC from DDTAVV motif of bacterial A. acidocaldarius SC caused six species: rat, Rattus rattus OSC; yeast, S. cerevisiae OSC; Candida, C. albicans OSC; plant, A. thaliana OSC (cycloartenol synthase); almost complete loss of cyclase activity.t bacteria-a, A. acidocaldarius SC; bacteria-z, Z. mobilis SC. Frequently The 2.8-kbp rat OSC cDNA was cloned into the yeast/E. coli occurring residues are in boldface; hyphens indicate gaps introduced shuttle vector pYEUra3 (Clontech) and functionally expressed to maximize alignment. in the OSC-deficient yeast mutant strain SGL9 (erg7 ura3-52 hem3-6 gal2) (16) under the control of the GALI promoter. microsomal cyclases appear to bind loosely to membranes; this Recent cloning of yeast and plant OSCs (15-20) were all may explain the ease of solubilization under mild conditions. achieved by complementation of this OSC-deficient (erg7) A similar situation was reported for the recently cloned rat mutant strain (33). The low copy number centromeric plasmid squalene epoxidase (47), another membrane-associated en- pYEUra3 is a modified version of AYES (pSE937) (48), which zyme that catalyzes formation of 2,3-oxidosqualene, the sub- was successfully used for expression of yeast OSC in the SGL9 strate for OSC. Furthermore, in rat OSC, there are five strain by Griffin and co-workers (16). The GAL] promoter is possible N-glycosylation sites (N-383, N-517, N-606, N-692, induced >1000-fold by the presence of galactose but is re- and N-698). Although preliminary results showed evidence for pressed by the presence of glucose (49). Like other OSC genes, protein glycosylation, incubation with N-glycosidase did not the rat OSC cDNA appeared to complement the erg7 defi- reduce OSC activity (unpublished results). ciency of the yeast mutant strain, since the transformant cells We reported previously that the two adjacent Asp residues could grow on media lacking ergosterol. (D-456 and D-457) in a putative DDTAEA motif (see Fig. 2) Enzyme activity for conversion of (3S)-2,3-[14C]oxidosqua- were labeled with the mechanism-based irreversible inhibitor lene to lanosterol was detected only after galactose induction; [3H]29-MOS (26, 27). However, according to the deduced the activity (27 fmol of lanosterol per min per mg of cell pellet) was <10% of wild-type yeast cyclase activity. A similar low amino acid sequence, it now appears that the 29-MOS binding OSC activity was found for heterologous expression of Can- site sequence just N-terminal of the QW-4 motif is actually dida OSC in the yeast erg7 strain (17). In contrast, plant A. DCTAEA, instead of DDTAEA. Interestingly, this sequence is well-conserved in all thaliana cycloartenol synthase OSC was successfully expressed the known OSCs (15, 16) despite the in the erg7 strain under the control of PGK1 promoter (20). fact that neither yeast nor plant OSCs can be labeled with The low specific activity of the recombinant rat OSC may be [3H]29-MOS (8). In Edman sequencing of the labeled peptide, due to decreased stability of the heterologous protein; after elution of the first steroid-modified Asp phenylthiohydantoin galactose induction, no novel protein band was observable at derivative might have carried over to the next cycle, leading to '80 kDa on SDS/PAGE. miscalling of the residue. This carryover would be particularly As a critical test of the authenticity of this heterologously difficult to detect given that it was followed by an underivatized expressed recombinant rat OSC, the cell-free extract was Cys. Therefore, only the Asp residue of the DCTAEA motif incubated with [3H]29-MOS, a mechanism-based inhibitor that was actually labeled with the suicide substrate. Site-directed mutagenesis experiments will provide a further test for this lPoralla, K., Ochs, D. & Feil, C., American Oil Chemists' Society hypothesis. Recently, Poralla and co-workers reported that a Symposium on the Regulation of Biosynthesis and Function of point mutation of the first Asp residue of the corresponding Isopentenoids, May 9-10, 1994, Atlanta. Downloaded by guest on October 2, 2021 9278 Biochemistry: Abe and Prestwich Proc. Natl. Acad. Sci. USA 92 (1995) A B 9. Moore, W. R. & Schatzman, G. L. (1992) J. Biol. Chem. 267, 2 3 4 4' 1 2 3 4 4' 22003-22006. kDa 10. Abe, I., Ebizuka, Y. & Sankawa, U. (1988) Chem. Pharm. Bull. 212, 36, 5031-5034. 158- 11. Abe, I., Sankawa, U. & Ebizuka, Y. (1989) Chem. Pharm. Bull. 116 - 37, 536-538. 97- 12. Abe, I., Ebizuka, Y., Seo, S. & Sankawa, U. (1989) FEBS Lett. 66- 249, 100-104. 56- 13. Abe, I., Sankawa, U. & Ebizuka, Y. (1992) Chem. Pharm. Bull. 40, 1755-1760. 43 14. Corey, E. J. & Matsuda, S. P. T. (1991) J. Am. Chem. Soc. 113, 37. 27- 8172-8174. 15. Corey, E. J., Matsuda, S. P. T. & Bartel, B. (1994) Proc. Natl. FIG. 5. [3H]29-MOS affinity labeling of cell-free extracts from the Acad. Sci. USA 91, 2211-2215. transformed yeast mutant strain SGL9 (erg7). Rat OSC cDNA was 16. Shi, Z., Buntel, C. & Griffin, J. H. (1994) Proc. Natl. Acad. Sci. expressed under the control ofyeast GAL] promoter. (A) SDS/PAGE USA 91, 7370-7374. (7.5%) gel stained with Coomassie blue. (B) Corresponding fluoro- 17. Kelly, R., Miller, S. M., Lai, M. H. & Kirsch, D. R. (1990) Gene gram. Lanes contain cell-free extracts as follows: lane 1, SGL9; lane 87, 177-183. 2, transformed SGL9 before galactose induction; lane 3, transformed 18. Buntel, C. J. & Griffin, J. H. (1992) J. Am. Chem. Soc. 114, SGL9 after induction; lane 4, purified rat native OSC loaded to 9711-9713. visualize stained protein; lane 4', purified rat native OSC loaded with 19. Roessner, C. A., Min, C., Hardin, S. H., Harris-Haller, L. W., enzyme activity equivalent to that of SGL9 extract in lane 3. McCollum, J. C. & Scott, A. I. (1993) Gene 127, 149-150. 20. Corey, E. J., Matsuda, S. P. T. & Bartel, B. (1993) Proc. Natl. forms a covalent linkage to an Asp residue of the vertebrate Acad. Sci. USA 90, 11628-11632. but not plant or yeast cyclase active site (8, 26). Fig. 5 shows 21. Ochs, D., Kaletta, C., Entian, K.-D., Beck-Sickinger, A. & a single radioactive band with the same molecular size ("80 Poralla, K. (1992) J. Bacteriol. 174, 298-302. kDa) as the native rat OSC. At equivalent levels of OSC 22. Reipen, I., Poralla, K., Sahm, H. & Sprenger, G. (1995) Micro- biology (Reading, UK) 141, 155-161. enzymatic activity, the intensity of labeling of the yeast- 23. Poralla, K., Hewelt, A., Prestwich, G. D., Abe, I., Reipen, I. & expressed recombinant rat OSC by [3H]29-MOS is essentially Sprenger, G. (1994) Trends Biochem. Sci. 19, 157-158. identical to that of the native rat enzyme. This report thus 24. Poralla, K. (1994) Bioorg. Med. Chem. Lett. 4, 285-290. constitutes isolation, sequencing, and functional expression of 25. Xiao, X.-y. & Prestwich, G. D. (1991) J. Am. Chem. Soc. 13, a vertebrate enzyme responsible for cyclization of 2,3- 9673-9674. oxidosqualene to lanosterol. 26. Abe, I. & Prestwich, G. D. (1994) J. Biol. Chem. 269, 802-804. 27. Abe, I. & Prestwich, G. D. (1995) Lipids 30, 231-234. Note. After the cDNA sequencing was completed,§ a short commu- 28. Bai, M. (1991) Ph.D. dissertation (State University of New York, nication describing the sequence of a cDNA encoding rat liver OSC Stony Brook). appeared (50). The communication, which does not provide any 29. Chomczynski, P. & Sacchi, N. (1987)Anal. Biochem. 162, 156-159. experimental details, shows a sequence (GenBank accession no. 30. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. D45252) that differs from that shown in Fig. 2 by seven amino acid Sci. USA 74, 5463-5467. residues; moreover, expression of a functional OSC protein was not 31. Towner, P. & Gartner, W. (1992) Nucleic Acids Res. 20, 4669- reported. 4670. 32. Zeiner, M. & Gehring, U. (1994) BioTechniques 17, 1050-1054. 33. Gollub, E. G., Liu, K.-p., Dayan, J., Adlersberg, M. & Sprinson, §Abe, I. & Prestwich, G. D., Poster Session, 14th Enzyme Mechanism D. B. (1977) J. Biol. Chem. 252, 2846-2854. Conference, January 4-7, 1995, Scottsdale, AZ. 34. Schiestl, R. H. & Gietz, R. D. (1989) Curr. Genet. 16, 339-346. 35. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8148. We are indebted to Professor John H. Griffin at Stanford University 36. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids for the gift of yeast mutant strain SGL9 and for his advice on cDNA Res. 12, 387-395. expression. We thank Mr. Thomas Fischer (Center of Analysis and 37. Kumpf, R. A. & Dougherty, D. A. (1993) Science 261,1708-1711. Synthesis of Macromolecules, Stony Brook) for Edman peptide se- 38. Johnson, W. S., Lindell, S. D. & Steele, J. (1987) J. Am. Chem. quencing and Ms. Brenda A. Madden for synthesis of (3S)-2,3- Soc. 109, 5852-5853. 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Chim. Acta 38, 1890-1904. 45. Post, B. & Sander, C. (1994) Proteins 19, 55-72. 4. van Tamelen, E. E. (1982) J. Am. Chem. Soc. 104, 6480-6481. 46. Post, B., Casadio, R., Fariselli, P. & Sander, C. (1995) Protein Sci. 5. Corey, E. J., Virgil, S. S. & Sarshar, S. (1991) J. Am. Chem. Soc. 4, 521-533. 113, 8171-8172. 47. Sakakibara, J., Watanabe, R., Kanai, Y. & Ono, T. (1995)J. Biol. 6. Corey, E. J. & Virgil, S. C. (1991) J. Am. Chem. Soc. 113, Chem. 270, 17-20. 4025-4026. 48. Ramer, S. W., Elledge, S. J. & Davis, R. W. (1992) Proc. Natl. 7. Kusano, M., Abe, I., Sankawa, U. & Ebizuka, Y. (1991) Chem. Acad. Sci. USA 89, 11589-11593. Pharm. Bull. 39, 239-241. 49. Johnston, M. & Davis, R. W. (1984) Mol. Cell. Biol. 4, 1440-1448. 8. Abe, I., Bai, M., Xiao, X.-y. & Prestwich, G. D. (1992) Biochem. 50. Kusano, M., Shibuya, M., Sankawa, U. & Ebizuka, Y. (1995) Biol. Biophys. Res. Commun. 187, 32-38. Pharm. Bull. 18, 195-197. Downloaded by guest on October 2, 2021