Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity.
3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40 degrees C and 7.5-8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0-7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.
Choi
Yun Hee
YH
Division of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Republic of Korea.
Choi
Hye Jeong
HJ
Kim
Dooil
D
Uhm
Ki-Nam
KN
Kim
Hyung-Kwoun
HK
D001665:Binding Sites; D004795:Enzyme Stability; D006843:Hydrocarbons, Chlorinated / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020005:Propanols / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
C507643:3-chloro-1-phenylpropanol; D006843:Hydrocarbons, Chlorinated; D020005:Propanols; D029701:Saccharomyces cerevisiae Proteins; D009249:NADP; D010088:Oxidoreductases; C432953:GRE2 protein, S cerevisiae