Mechanism-guided remodeling of a push-pull active-site microenvironment in aldehyde dehydrogenase for HMF oxidation.

Abstract

Engineering enzyme microenvironments for selective oxidation of biomass-derived aldehydes remains challenging because productive substrate positioning must be achieved within an active site that also accommodates a chemically distinct carboxylic acid product. Here, we report mechanism-guided engineering of an NAD+-dependent aldehyde dehydrogenase from Shigella dysenteriae (SdALDH) for selective oxidation of 5-hydroxymethylfurfural (HMF) to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). Structure-guided analysis of the active-site pocket identified V301C as a beneficial substitution, which was subsequently used as the starting variant for pH-responsive screening and led to the identification of the additional L173I substitution. The resulting double variant V301C-L173I afforded 85.2% HMF conversion and 73.2% HMFCA yield under standardized whole-cell conditions. Its catalytic efficiency increased from 2.23 to 12.57 mM-1 s-1, corresponding to an approximately 464% increase relative to WT SdALDH. Comparative kinetic analysis further showed that V301C and L173I make distinct but complementary contributions to the overall catalytic improvement. Substrate-associated molecular simulations support a proposed push-pull model in which polar anchoring near residue 301 and local packing changes around residue 173 favor productive HMF positioning relative to catalytic C302, whereas HMFCA-associated simulations reveal reorganization of the local polar-contact network in the engineered active site. At the process level, coupling V301C-L173I with NOX-mediated NAD+ regeneration and dynamic pH-stat control enabled an endpoint HMFCA yield of 99.3% from 300 mM HMF after 18 h. These results demonstrate how coordinated active-site engineering and subsequent process intensification can improve SdALDH-mediated HMF oxidation while maintaining a clear distinction between enzyme-level and reaction-level contributions.

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