期刊
ACS CATALYSIS
卷 -, 期 -, 页码 -出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.3c03769
关键词
bioelectrocatalysis; direct electron transfer; cryo-electron microscopy; membrane-bound d-fructosedehydrogenase; intramolecular electron transfer
In this study, the flavin adenine dinucleotide-dependent d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 was investigated using structural biology, bioelectrochemistry, and protein engineering. The FDH showed intense direct electron transfer (DET)-type catalytic activity, making it a promising enzyme for biosensors. Structural analysis and mutagenesis experiments revealed the electron transfer pathway and the key residues involved in the DET-type activity of FDH.
Flavin adenine dinucleotide-dependent d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of direct electron transfer (DET)-type bioelectrocatalysis, was investigated from the perspective of structural biology, bioelectrochemistry, and protein engineering. DET-type reactions offer several benefits in biomimetics (e.g., biofuel cells, bioreactors, and biosensors) owing to their mediator-less configuration. FDH provides an intense DET-type catalytic signal; therefore, extensive research has been conducted on the fundamental principles and applications of biosensors. Structural analysis using cryo-electron microscopy and single-particle analysis has revealed the entire FDH structures with resolutions of 2.5 and 2.7 & Aring; for the reduced and oxidized forms, respectively. The electron transfer (ET) pathway during the catalytic oxidation of d-fructose was investigated by using both thermodynamic and kinetic approaches. Structural analysis has shown the localization of the electrostatic surface charges around heme 2c in subunit II, and experiments using functionalized electrodes with a controlled surface charge support the notion that heme 2c is the electrode-active site. Furthermore, two aromatic amino acid residues (Trp427 and Phe489) were located in a possible long-range ET pathway between heme 2c and the electrode. Two variants (W427A and F489A) were obtained by site-directed mutagenesis, and their effects on DET-type activity were elucidated. The results have shown that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET according to bioelectrochemical analysis.
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