title: A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression.
abstract: A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol min(-1) mg(-1), and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.
authors.affiliation: Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. mitukura@gifu-u.ac.jp
authors.forename: Koichi
authors.initials: K
authors.lastname: Mitsukura
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Kuramoto
authors.forename: Toyokazu
authors.initials: T
authors.lastname: Yoshida
authors.forename: Norihiro
authors.initials: N
authors.lastname: Kimoto
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Toru
authors.initials: T
authors.lastname: Nagasawa
mesh_terms: D000588:Amines / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D007097:Imines / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D000588:Amines; D001426:Bacterial Proteins; D007097:Imines; D009249:NADP; D010088:Oxidoreductases