Engineering the α- and β-subunit interface of a coenzyme B12-dependent glycerol dehydratase for enhancing its resistance to inactivation.

Glycerol dehydratase (GDHt) enables bioconversion of glycerol to valuable chemicals, but its industrial use is hindered by rapid loss of the adenosylcobalamin (AdoCbl) cofactor (coenzyme B12) through both oxygen- and mechanism-based inactivation. To overcome this limitation, we reinforced the AdoCbl-binding interface of Klebsiella pneumoniae GDHt by fusing its α and β subunits with a five-residue linker (fGDHt) and then introducing interface mutations. Fusion alone doubled the oxygen-inactivation half-life without affecting catalytic efficiency. Structural and computational analyses of interface residues, followed by experimental screening, yielded three stabilizing substitutions-α-A177M, β-L113W, and β-M158W. Pairwise combinations of these mutations yielded double variants whose oxygen-inactivation half-lives increased by up to 24-fold. Enzyme-coupled reactions to convert glycerol into 3-hydroxypropionic acid (3-HP) confirmed that engineered fGDHt variants maintained catalytic activity for longer periods, implying protection against both inactivation modes. In recombinant Escherichia coli strains producing 3-HP, the α-A177M/β-M158W variant matched wild-type titers while operating with 25-fold less AdoCbl. Crystal structures reveal that the mutations tighten inter-subunit packing and, in the case of α-A177M, partly occlude an O2-access tunnel to the cofactor. These results have established α-β interface engineering as a strategy for engineering more robust GDHts.