Cloning and overexpression of the Exiguobacterium sp. F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate.
Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho, Fukui 910-1195, Japan. wada@chem.agr.hokudai.ac.jp
Masaru
M
Wada
Ayumi
A
Yoshizumi
Yuki
Y
Furukawa
Hiroshi
H
Kawabata
Makoto
M
Ueda
Hiroshi
H
Takagi
Shigeru
S
Nakamori
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011422:Propionates / Q000378:metabolism*; D013876:Thiophenes / Q000378:metabolism*
D004269:DNA, Bacterial; D011422:Propionates; D013876:Thiophenes; D010088:Oxidoreductases