Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

Abstract

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme’s catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound d-Fructose Dehydrogenase with Structural Bioelectrochemistry

“… The site-directed mutagenesis and electron paramagnetic resonance (EPR) spectrum has indicated that three Cys residues in the Cys-rich region in the large subunit constitute the 3Fe-4S cluster (3Fe4S), which is thought to mediate the ET between FAD and heme c moieties . Another partial 3D structure composed of large and small subunits and a truncated cytochrome subunit, leaving only one heme c moiety ( Bc GDHγα-trβ), was also elucidated (PDB ID: 8HDD) to support this discussion . However, the overall structure of the membrane-bound subunit, including heme c moieties responsible for the electrode-active site, remains unknown.…”

“…42 Another partial 3D structure composed of large and small subunits and a truncated cytochrome subunit, leaving only one heme c moiety (BcGDHγα-trβ), was also elucidated (PDB ID: 8HDD) to support this discussion. 43 However, the overall structure of the membrane-bound subunit, including heme c moieties responsible for the electrode-active site, remains unknown. FDH is a particular enzyme with intense DET-type bioelectrocatalytic activity and has been extensively investigated from electrochemistry, protein engineering, and spectroscopy perspectives.…”