Yashima, Y. | Search bioRxiv

bioRxiv2026

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

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.

biochemistry↗

bioRxiv2018

Effect of continuous compressive force on the expression of RANKL, OPG, and VEGF in MC3T3-E1 and MLO-Y4 cells

Osteocytes, known to have mechano-sensory functions, influence the regulation of bone remodeling. However, the mechanism by which osteocytes regulate bone metabolism when mechanical forces are being applied is still unclear. Osteoclastogenesis is mainly regulated by receptor activator of nuclear factor kappa-B ligand (RANKL); the protein osteoprotegerin (OPG) and angiogenesis also play important roles in osteogenesis. RANKL, OPG, and vascular endothelial growth factor (VEGF) are thought to be key factors for bone metabolism. In this study, we examined the effect of a continuous compressive force (CF) on the expression of RANKL, OPG, and VEGF in osteoblastic murine osteocytes (MLO-Y4) and osteoblastic (MC3T3-E1) cells. Gene and protein expression levels of RANKL, OPG, and VEGF in MLO-Y4 and MC3T3-E1 cells were quantitatively determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Both cell types were also subjected to a CF of 1.0 g/cm2 for 1, 3, 6, and 12 hours. Furthermore, the effect of a stretch-activated (S-A) channel was examined by gadolinium (Gd3+) administration. The ratio of gene and protein expressions of RANKL, VEGF, and RANKL/OPG in MLO-Y4 cells were significantly higher than in MC3T3-E1 cells, while the expression of OPG was significantly lower. After CF application, both cell types showed significant increases in RANKL and VEGF expression as well as the RANKL/OPG ratio. Additionally, the upregulated gene and protein levels of these factors were reduced by Gd3+ administration.\n\nThese findings suggest that osteocytes play more important roles in bone metabolism and angiogenesis than osteoblasts. Osteocytes regulate the expression of RANKL, OPG, and VEGF via the S-A channel through the response to mechanical stress.

cell biology↗