9VIJ
Crystal structure of fused glycerol dehydratase A177M/M158W variant
Summary for 9VIJ
| Entry DOI | 10.2210/pdb9vij/pdb |
| Descriptor | Glycerol dehydrase alpha subunit,Glycerol dehydrase beta subunit, Glycerol dehydrase gamma subunit, COBALAMIN (3 entities in total) |
| Functional Keywords | glycerol dehydratase, lyase |
| Biological source | Klebsiella pneumoniae More |
| Total number of polymer chains | 4 |
| Total formula weight | 200044.73 |
| Authors | |
| Primary citation | Na, C.Y.,Nasir, A.,Park, R.,Yeon, Y.J.,Baek, S.H.,Park, S.,Park, Y.S.,Seo, M.D.,Yoo, T.H. Engineering the alpha- and beta-subunit interface of a coenzyme B 12 -dependent glycerol dehydratase for enhancing its resistance to inactivation. Bioresour Technol, 442:133733-133733, 2026 Cited by PubMed Abstract: 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 B) 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 O-access tunnel to the cofactor. These results have established α-β interface engineering as a strategy for engineering more robust GDHts. PubMed: 41319883DOI: 10.1016/j.biortech.2025.133733 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (3.58 Å) |
Structure validation
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