Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

• Published in: Chemistry : a European journal Vol. 23; no. 51; pp. 12636 - 12645
• Main Authors: Ensari, Yunus, Dhoke, Gaurao V., Davari, Mehdi D., Bocola, Marco, Ruff, Anna Joëlle, Schwaneberg, Ulrich
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 12-09-2017
• Subjects: Alcohol; Alcohol dehydrogenase; Alcohols; Aliphatic compounds; Chains; Chemistry; directed evolution; Enzymes; fatty acid methyl ester; Fatty acids; Libraries; methyl 3-hydroxyalkanoate; Molecular docking; Oxidation; protein engineering; Substrates; Synthesis; methyl 3-hydroxyalkanoate; fatty acid methyl ester; protein engineering; alcohol dehydrogenase; directed evolution
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201702581

Expanding the substrate scope of enzymes opens up new routes for synthesis of valuable chemicals. Ketone‐functionalized fatty acid derivatives and corresponding chiral alcohols are valuable building blocks for the synthesis of a variety of chemicals including pharmaceuticals. The alcohol dehydrogenase from Candida parapsilosis (cpADH5) catalyzes the reversible oxidations of chiral alcohols and has a broad substrate range; a challenge for cpADH5 is to convert alcohols with small substituents (methyl or ethyl) next to the oxidized alcohol moiety. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In the current manuscript, we report that positions L119 and W286 are key residues to boost oxidation of medium chain methyl 3‐hydroxy fatty acids; interestingly the enantiopreference toward methyl 3‐hydroxybutyrate was inverted. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3‐hydroxyhexanoate (Vmax: 2.48 U mg− and Km: 4.76 mm). Simultaneous saturation at positions 119 and 286 library yielded a double mutant (L119M/W286S) with more than 30‐fold improved activity toward methyl 3‐hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %) and inverted enantiopreference (S‐enantiomer ≥99 % activity decrease and R‐enantiomer >20‐fold activity improvement) toward methyl 3‐hydroxybutyrate.
cpADH5 WT is a S‐selective alcohol dehydrogenase (ADH) and does not convert 3‐hydroxy fatty acid methyl esters larger than methyl 3‐hydroxypentanoate. Thus, methyl 3‐hydroxyhexanoate and methyl (R)‐3‐hydroxybutyrate are not accepted as substrate by cpADH5 WT. W286A substitution led to chain length specificity shift and oxidation of methyl 3‐hydroxyhexanoate to corresponding ketone. Furthermore, L119M/W286S variant showed enantiopreference inversion for methyl 3‐hydroxybutyrate from S‐enantiomer to R‐enantiomer.