Molecular Cloning and Expression of Rat Squalene Epoxidase (*)
- From the (1)Department of Biochemistry, Niigata University School of Medicine, Niigata 951 and the
- (2)Department of Agricultural Chemistry, Faculty of Agriculture, Niigata University, Niigata 950-21, Japan
- § To whom correspondence should be addressed: Dept. of Biochemistry, Niigata University School of Medicine, 1-757 Asahimachidori, Niigata 951, Japan. Tel.: 81-25-223-6161; Fax: 81-25-222-4599.
Abstract
Squalene epoxidase (SE) (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be
one of the rate-limiting enzymes in this pathway. Rat SE cDNA was isolated by selecting yeast transformants expressing rat
cDNA in the presence of terbinafine, an inhibitor specific for fungal SE. The expression of rat SE in the isolated terbinafine-resistant
clone was confirmed by its survival in the presence of either terbinafine or an inhibitor specific for mammalian SE, NB-598,
but not in the presence of both terbinafine and NB-598. Rat SE polypeptide deduced from the nucleotide sequence contains 573
amino acids, and its molecular weight is 63,950 Da. The amino acid sequence reveals one potential transmembrane domain, a
hydrophobic segment (Leu
to Tyr
) in the NH2-terminal region. This region also contains a β1-αA-β2 motif, which is the consensus sequence for an FAD binding domain, suggesting
that SE is a flavoenzyme. This deduced rat SE sequence is 30.2% identical to the ERG 1 gene, which encodes SE from an allylamine-resistant Saccharomycescerevisiae mutant. Expression of a full-length rat SE protein in Escherichia coli confirms this polypeptide as a functional SE. This is the first report of the molecular cloning of mammalian SE.
Footnotes
-
↵* This work was supported by Grants-in-aid 06770080 and 06557135 for Scientific Research from the Ministry of Education, Science and Culture, Japan. Financial support was also provided by the Cosmetology Research Foundation, by ONO Medical Research Foundation, and by the Chiyoda Mutual Life Foundation, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) D37920[GenBank].
-
↵1 The abbreviations used are:
- SE
-
squalene epoxidase
- PCR
-
polymerase chain reaction
- DTT
-
dithiothreitol
- bp
-
base pair(s).
-
- Received August 25, 1994.
- Revision received October 26, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
