Robust NADH-regenerator: improved alpha-haloketone-resistant formate dehydrogenase

Applied Microbiology and Biotechnology
H YamamotoN Esaki

Abstract

Formate dehydrogenases (FDH) are useful for the regeneration of NADH, which is required for asymmetric reduction by several dehydrogenases and reductases. FDHs have relatively low activity and are labile, especially to alpha-haloketones, thus FDH cannot be applied to the industrial manufacture of optically active alpha-haloalcohols. To stabilize a FDH from Mycobacterium vaccae (McFDH) against the alpha-haloketone ethyl 4-chloroacetoacetate (ECAA), a set of cysteine-mutant enzymes was constructed. Sensitivity to ECAA of mutant C6S was similar to that of the wild-type enzyme, and mutants C249S and C355S showed little activity. In contrast, mutant C256S exhibited remarkable tolerance to ECAA. Surprisingly, mutant C146S was activated by several organic compounds such as ethyl acetate. An optimized mutant, C6A/C146S/C256V (McFDH-26), was obtained by combining several effective mutations. Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-ECHB] was synthesized from ECAA to 49.9 g/l with an optical purity of more than 99% e.e. using recombinant Escherichia coli cells coexpressing McFDH-26 and a carbonyl reductase (KaCR1) from Kluyveromyces aestuarii.

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Citations

May 17, 2012·Applied Microbiology and Biotechnology·Kathrin HoelschDirk Weuster-Botz
Jul 2, 2010·Biotechnology and Applied Biochemistry·Kathrin Hölsch, Dirk Weuster-Botz
Dec 6, 2014·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Luisa B MaiaIsabel Moura
May 26, 2007·Protein Science : a Publication of the Protein Society·Katja SchirwitzVictor S Lamzin
Sep 3, 2010·Journal of Applied Toxicology : JAT·Cécile TrinhLuc Verschaeve
Jul 6, 2019·Frontiers in Bioengineering and Biotechnology·Rajendran Velmurugan, Aran Incharoensakdi
Mar 21, 2006·Biomolecular Engineering·Vladimir I Tishkov, Vladimir O Popov
Aug 1, 2020·ACS Catalysis·Liliana Calzadiaz-RamirezCarlos G Acevedo-Rocha
Oct 12, 2021·Critical Reviews in Biotechnology·Saadet AlpdağtaşBarış Binay

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