Structural basis for loss of covalent flavinylation in the H158Y mutant of pyranose oxidase

Abstract

Pyranose oxidase from Phanerochaete chrysosporium (PcPOx) is a flavoprotein that forms a covalent 8-N3-histidyl-FAD linkage at His158. Here, we determined the crystal structure of the PcPOx H158Y variant at 1.69 [A] resolution and investigated its FAD occupancy and catalytic activity. Tyr158 adopts a rotamer conformation incompatible with covalent flavinylation, placing its phenolic O atom 8.7 [A] away from the FAD C8 atom. This conformation is stabilized through a Tyr158-Lys79 hydrogen bond. The additional mutation K79A abolishes this hydrogen bond, potentially freeing the Tyr158 residue for other conformations, but does not restore covalent flavin attachment. The H158Y variant retains substantial FAD occupancy and tetrameric assembly but shows markedly reduced oxidase and dehydrogenase turnover rates (less than 13% of WT). These results provide a structural explanation for the failure of tyrosine substitution to support covalent flavinylation in PcPOx and offer insights into requirements for engineering alternative covalent flavin linkages.