known oxidosqualene cyclases. This enzyme family converts
oxidosqualene to cyclic triterpene alcohols that are the
precursors to membrane sterols. Sequencing revealed that
G14J10 could encode a 300 amino acid fragment similar to
the N-termini of known oxidosqualene cyclases. In pursuit
of a full-length clone, the fragment was radiolabeled with
[R-32P]-dCTP and was used to screen a λgt11 T. cruzi CL
strain epimastigote genomic library8 using established pro-
tocols.9 Although nine of ∼6 × 105 plaques hybridized
strongly, no full-length clone was obtained. However,
sequencing the longest clone (Genbank accession number
AF285827) revealed that it contained the missing 3′ se-
quence, and it lacked only 130 bp from the putative start
site. Attempts to PCR-amplify the 5′ end of the gene from
this genomic DNA library using G14J10 clone-specific
primers were unsuccessful, as were attempts to amplify the
complete coding sequence from a cDNA pool using spliced
leader-specific primers. Fortunately, both fragments share a
unique BamH I site, which was used to subclone the two
together and reassemble the complete open reading frame.
indicating that the compound obtained was a product of the
recombinant T. cruzi oxidosqualene cyclase.
NMR and gas chromatographic studies showed that the
1
product was lanosterol. A 400 MHz H NMR spectrum
contained key signals that are within 0.01 ppm of charac-
teristic lanosterol signals:15 δ 0.688 (C-18, s, 3 H), 0.810
(C-29, s, 3 H), 0.873 (C-30, s, 3 H), 0.911 (C-21, d, J ) 6.4
Hz, 3 H), 0.981 (C-19, s, 3 H), 1.000 (C-28, s, 3 H), 1.601
(C-27, s, 3 H), 1.681 (C-26, s, 3 H), 3.23 (C-3, dd, J ) 3.8,
11.4, 3 H), and 5.10 ppm (C-24, m, 1 H). The alcohol product
was acetylated with pyridine/acetic anhydride (1:1). Gas
chromatography showed a single signal that comigrated with
lanosteryl acetate (retention time of standard relative to that
of cholesteryl acetate ) 1.332; that of sample to cholesteryl
acetate ) 1.325). Neither cycloartenyl acetate nor parkeyl
acetate was observed (<0.5% detection limit).
These in vivo complementation and in vitro product
analysis experiments establish that T. cruzi encodes a
lanosterol synthase (Scheme 1). Animals and fungi biosyn-
thesize sterol from lanosterol, whereas plants biosynthesize
structurally similar sterols from cycloartenol. Difficulties in
large-scale culture have precluded classical metabolic studies
to establish which route T. cruzi uses. The existence of a T.
cruzi lanosterol synthase establishes that lanosterol is a T.
cruzi metabolite and is consistent with this pathogen using
lanosterol as a sterol biosynthetic intermediate. A similar
recombinant expression approach established lanosterol as
a T. brucei metabolite,13 and lanosterol biosynthesis has been
demonstrated16 in the kinetoplastid Crithidia fasciculata.
Thus, kinetoplastids apparently utilize a different initial cyclic
intermediate from most other protists (including the amoebae
Acanthamoeba polyphaga,17 Dictyostelium discoideum,18
Naegleria loVaniensis and N. gruberi,19 and the euglenids
Euglena gracilis20 and Astasia longa21), which biosynthesize
sterol from cycloartenol as plants do.
For complementation studies, the reconstructed coding
sequence was subcloned into the galactose-inducible integra-
tive yeast expression vector pRS305GAL10 and the high-
copy derivative pRS426GAL11 to construct pBJ1.21 and
pBJ1.22, respectively. Lithium acetate was used to transform9
the Saccharomyces cereVisiae lanosterol synthase mutant
SMY810 with both constructs. When plated on expression
medium (1% yeast extract, 2% peptone, 2% galactose, 20
mg/L heme) lacking ergosterol, strains expressing the T. cruzi
oxidosqualene cyclase grew similarly to a positive control
(SMY8 expressing native yeast lanosterol synthase from the
same plasmid).12 A negative control (SMY8 with empty
vector) required ergosterol to grow. These experiments show
that the T. cruzi oxidosqualene cyclase gene genetically
complements the yeast lanosterol synthase mutant, suggesting
that it encodes a lanosterol synthase.
The reconstructed 2.7 kbp T. cruzi lanosterol synthase gene
encodes a predicted 902 amino acid protein that is 67%
identical to that of T. brucei,13 28-35% identical to other
known lanosterol synthases,10,12,22 and 30-32% identical to
The high-copy construct was used to transform the
squalene synthase-lanosterol synthase double mutant LHY411
to analyze the enzymatic properties in vitro. An extract
prepared from a 1-L culture (8 g of yeast) of the LHY4-
[pBJ1.22] transformant was incubated13 with 20 mg (0.5 mg/
mL) of racemic oxidosqualene.14 After 24 h at 25 °C, the
reaction was quenched with 2 volumes of ethanol, and
product was isolated essentially as described.11 Column
chromatography (5:1 hexane/methylene chloride) provided
8.2 mg of product (82% yield). The control yeast strain
containing empty vector did not cyclize oxidosqualene,
(15) Emmons, G. T.; Wilson, W. K.; Schroepfer, G. J. Jr. Magn. Reson.
Chem. 1989, 27, 1012-1024.
(16) Raederstorff, D.; Rohmer, R. FEMS Microbiol. Lett. 1986, 34, 269-
272.
(17) Raederstorff, D.; Rohmer, M. Biochem. J. 1985, 231, 609-615.
(18) (a) Nes, W. D.; Norton, R. A.; Crumley, F. G.; Madigan, S. J.;
Katz, E. R. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 7565-7569. (b) Godzina,
S. M.; Lovato, M. A.; Meyer, M. M.; Foster, K. A.; Wilson, W. K.; Gu,
W.; de Hostos, E. L.; Matsuda, S. P. T. Lipids 2000, 36, 249-255.
(19) Raederstorff, D.; Rohmer, M. Eur. J. Biochem. 1987, 164, 427-
434.
(8) Van Voorhis, W. C.; Eisen, H. J. Exp. Med. 1989, 169, 641-652.
(9) Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidman,
J. G.; Smith, J. A.; Struhl, K. Current Protocols in Molecular Biology;
Wiley-Interscience: New York, 1999.
(20) Anding, C.; Brandt, R. D.; Ourisson, G. Eur. J. Biochem. 1971, 24,
259-263.
(21) Rohmer, M.; Brandt, R. D. Eur. J. Biochem. 1973, 36, 446-454.
(22) (a) Buntel, C. J.; Griffin, J. H. J. Am. Chem. Soc. 1992, 114, 9711-
9713. (b) Roessner, C. A.; Min, C.; Hardin, S. H.; Harris-Haller, L. W.;
McCollum, J. C.; Scott, A. I. Gene 1993, 127, 149-150. (c) Shi, Z.; Buntel,
C. J.; Griffin, J. H. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 7370-7374. (d)
Kusano, M.; Shibuya, M.; Sankawa, U.; Ebizuka, Y. Biol. Pharm. Bull.
1995, 18, 195-197. (e) Abe, I.; Prestwich, G. D. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 9274-9278. (f) Baker, C. H.; Matsuda, S. P. T.; Liu, D.
R.; Corey, E. J. Biochem. Biophys. Res. Commun. 1995, 213, 154-160.
(g) Sung, C.-K.; Shibuya, M.; Sankawa, U.; Ebizuka, Y. Biol. Pharm. Bull.
1995, 18, 1459-1461.
(10) Corey, E. J.; Matsuda, S. P. T.; Baker, C. H.; Ting, A. Y.; Cheng,
H. Biochem. Biophys. Res. Commun. 1996, 219, 327-331.
(11) Hart, E. A.; Hua, L.; Darr, L. B.; Wilson, W. K.; Pang, J.; Matsuda,
S. P. T. J. Am. Chem. Soc. 1999, 121, 9887-9888.
(12) Corey, E. J.; Matsuda, S. P. T.; Bartel, B. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 2211-2215.
(13) Buckner, F. S.; Ngyuen, L. N.; Joubert, B. M.; Matsuda, S. P. T.
Mol. Biochem. Parasitol. 2000, 110, 399-403.
(14) Nadeau, R. G.; Hanzlik, R. P. Methods Enzymol. 1968, 15, 346-
351.
1958
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