title: Efficient synthesis of (R)-3-hydroxypentanenitrile in high enantiomeric excess by enzymatic reduction of 3-oxopentanenitrile.
abstract: (R)-3-Hydroxypentanenitrile (HPN) is an important intermediate in the synthesis of an immunosuppressive inosine 5′-monophosphate dehydrogenase inhibitor. An efficient enzymatic procedure for the synthesis of (R)-HPN with over 99 % enantiomeric excess using a novel acetoacetyl-CoA reductase (AdKR) from Achromobacter denitrificans was successfully established. Many microorganisms are known to reduce 3-oxopentannitrile (KPN) to (R)-HPN. An enzyme from A. denitrificans partially purified using ion exchange chromatography reduced KPN to (R)-HPN with high enantioselectivity. The AdKR gene was cloned and sequenced and found to comprise 738 bp and encode a polypeptide of 26,399 Da. The deduced amino acid sequence showed a high degree of similarity to those of other putative acetoacetyl-CoA reductases and putative 3-ketoacyl-ACP reductases. The AdKR gene was singly expressed and coexpressed together with a glucose dehydrogenase (GDH) as a coenzyme regenerator in Escherichia coli under the control of the lac promoter. (R)-HPN was synthesized with over 99 % e.e. using a cell-free extract of recombinant E. coli cells coexpressing AdKR and GDH.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kawano
authors.forename: Junzo
authors.initials: J
authors.lastname: Hasegawa
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Yasohara
mesh_terms: D042441:Achromobacter denitrificans / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009570:Nitriles / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D004269:DNA, Bacterial; D009570:Nitriles; D010088:Oxidoreductases