Chembiochem. 2026 Jun 15;27(11):e70426. doi: 10.1002/cbic.70426.

ABSTRACT

Formate dehydrogenase (FDH) catalyzes the reversible formate oxidation using NAD⁺ to yield carbon dioxide and NADH. FDH is widely utilized for cofactor regeneration, enabling the continuous supplying of NADH for chiral chemical biosynthesis. Herein, we employed semi-rational engineering to improve the thermostability of highly catalytic FDH from Bacillus simplex (BsFDH). Through two rounds of mutational screening, BsFDH^Q125F variant was identified to exhibit significantly improved thermostability with a 25-fold increase in half-life at 60°C. Additionally, the BsFDH^Q125F variant revealed significant stability against chemo-inactivation. The 100-ns molecular dynamics stimulation demonstrated a reduced overall root-mean-square deviation for the BsFDH^Q125F, with enhanced local packing primarily driven by intra- and inter-subunit π-π interaction among four tandem histidine and phenylalanine residues, thereby restricting the movement of flexible loop130-160. The implementation of the BsFDH^Q125F variant for cofactor regeneration in 4-nitrophenol detoxification demonstrated its superior stability and efficiency under bioconversion, rendering it suitable for various biocatalytic industrial applications.

PMID:42281337 | DOI:10.1002/cbic.70426