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

Autor: Dhoke GV; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany., Ensari Y; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.; Kafkas University, Faculty of Engineering and Architecture, Department of Bioengineering, full address?, Kars, Turkey., Hacibaloglu DY; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany., Gärtner A; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany., Ruff AJ; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany., Bocola M; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany., Davari MD; Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2020 Oct 15; Vol. 21 (20), pp. 2957-2965. Date of Electronic Publication: 2020 Jun 30.
DOI: 10.1002/cbic.202000247
Abstrakt: 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.
(© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)
Databáze: MEDLINE
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