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Title
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Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme.
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Authors
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T.Yoshimoto,
H.Higashi,
A.Kanatani,
X.S.Lin,
H.Nagai,
H.Oyama,
K.Kurazono,
D.Tsuru.
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Ref.
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J Bacteriol, 1991,
173,
2173-2179.
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PubMed id
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Abstract
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The 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) gene from Escherichia
coli HB101 was cloned and expressed in E. coli DH1. The hybrid plasmid pSD1,
with a 2.8-kbp insert of chromosomal DNA at the BamHI site of pBR322, was
subcloned into pUC19 to construct plasmid pSD3. The entire nucleotide sequence
of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy
chain-termination method. Within this sequence, the mature enzyme
protein-encoding sequence was found to start at a GTG initiation codon and to
comprise 765 bp, as judged by comparison with the protein sequence. The deduced
amino acid sequence of the enzyme indicated that the molecular weight is 26,778.
The transformant of E. coli DH1 harboring pSD3 with a 1.8-kbp fragment showed
about 200-fold-higher enzyme activity than the host. The enzyme was purified by
a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an
activity yield of 39%. The purified enzyme was homogeneous, as judged by sodium
dodecyl sulfate gel electrophoresis. The enzyme was most active at pH 8.5 and
stable between pH 8 and 9. The enzyme was NAD+ dependent and had a pI of 4.3.
The molecular mass was estimated to be 120 kDa by the gel filtration method and
28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric
form.
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