Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella

Michele Partipilo, Jacob J Whittaker, Nicola Pontillo, Jelmer Coenradij, Andreas Herrmann, Albert Guskov, Dirk Jan Slotboom*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD + ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (Fdh SNO ) from the soil bacterium Starkeya novella strictly specific for NAD + . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of Fdh SNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP + ) with better catalytic efficiency than NAD + . The single mutation D221Q enabled the reduction of NADP + with a catalytic efficiency k CAT /K M of 0.4 s -1 mM -1 at 200 mM formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a 5-fold increase in catalytic efficiency for NADP + compared to the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP + . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of Fdh SNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.

Original languageEnglish
Pages (from-to)4238-4255
Number of pages18
JournalThe FEBS Journal
Volume290
Issue number17
Early online date22-May-2023
DOIs
Publication statusPublished - Sept-2023

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