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NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.

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