Reversal of Regioselectivity in Zinc‐Dependent Medium‐Chain Alcohol Dehydrogenase from Rhodococcus erythropolis toward Octanone Derivatives

• Published in: Chembiochem : a European journal of chemical biology Vol. 21; no. 20; pp. 2957 - 2965
• Main Authors: Dhoke, Gaurao V., Ensari, Yunus, Hacibaloglu, Dinc Yasat, Gärtner, Anna, Ruff, Anna Joëlle, Bocola, Marco, Davari, Mehdi D.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 15.10.2020; John Wiley and Sons Inc
• Subjects: Alcohol dehydrogenase; alcohol dehydrogenases; Alcohols; Biocatalysts; Carbonyl compounds; Carbonyl groups; Carbonyls; Chains; Computer applications; Dehydrogenase; Dehydrogenases; Derivatives; homology modeling; Hypertension; Ketones; molecular docking; Phenylbutyric acid; Protein engineering; Regioselectivity; Rhodococcus; Rhodococcus erythropolis; Saturation mutagenesis; Stereoselectivity; Substrates; Synthesis; Zinc; Rhodococcus erythropolis; protein engineering; homology modeling; molecular docking; alcohol dehydrogenases
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.202000247

The zinc‐dependent medium‐chain alcohol dehydrogenase from Rhodococcus erythropolis (ReADH) is one of the most versatile biocatalysts for the stereoselective reduction of ketones to chiral alcohols. Despite its known broad substrate scope, ReADH only accepts carbonyl substrates with either a methyl or an ethyl group adjacent to the carbonyl moiety; this limits its use in the synthesis of the chiral alcohols that serve as a building blocks for pharmaceuticals. Protein engineering to expand the substrate scope of ReADH toward bulky substitutions next to carbonyl group (ethyl 2‐oxo‐4‐phenylbutyrate) opens up new routes in the synthesis of ethyl‐2‐hydroxy‐4‐phenylbutanoate, an important intermediate for anti‐hypertension drugs like enalaprilat and lisinopril. We have performed computer‐aided engineering of ReADH toward ethyl 2‐oxo‐4‐phenylbutyrate and octanone derivatives. W296, which is located in the small binding pocket of ReADH, sterically restricts the access of ethyl 2‐oxo‐4‐phenylbutyrate, octan‐3‐one or octan‐4‐one toward the catalytic zinc ion and thereby limits ReADH activity. Computational analysis was used to identify position W296 and site‐saturation mutagenesis (SSM) yielded an improved variant W296A with a 3.6‐fold improved activity toward ethyl 2‐oxo‐4‐phenylbutyrate when compared to WT ReADH (ReADH W296A: 17.10 U/mg and ReADH WT: 4.7 U/mg). In addition, the regioselectivity of ReADH W296A is shifted toward octanone substrates. ReADH W296A has a more than 16‐fold increased activity toward octan‐4‐one (ReADH W296A: 0.97 U/mg and ReADH WT: 0.06 U/mg) and a more than 30‐fold decreased activity toward octan‐2‐one (ReADH W296A: 0.23 U/mg and ReADH WT: 7.69 U/mg). Computational and experimental results revealed the role of position W296 in controlling the substrate scope and regiopreference of ReADH for a variety of carbonyl substrates. Reversal of regioselectivity: A versatile ADH from R. erythropolis (ReADH) accepts only carbonyl substrates with either a methyl or ethyl group adjacent to carbonyl moiety. Modified ReADH converts bulky substrates (ethyl 2‐oxo‐4‐phenylbutyrate) and opens up a new route for the synthesis of ethyl‐2‐hydroxy‐4‐phenylbutanoate, which is an important intermediate for anti‐hypertension drugs like enalaprilat and lisinopril.