Mechanistic studies of formate oxidase from Aspergillus oryzae: A novel member of the glucose-Methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids

• Published in: Archives of biochemistry and biophysics 2018-04, Vol.643, p.24-31
• Main Authors: Robbins, John M., Bommarius, Andreas S., Gadda, Giovanni
• Format: Article
• Language: English
• Subjects: 8-formyl FAD; Analytical ultracentrifugation; Formate oxidation; Isotope effects; pH effects; Viscosity effects
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2018.02.007

Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The kcat/Kformate value decreased from a value of 10,000 M−1s−1 at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the kcat/Kformate value indicated an isomerization of the Michaelis complex. The kcat/Koxygen value was 340,000 M−1s−1 and pH independent up to pH 6.0. The Dkcat and D(kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The appkred,H value was ∼75% that of kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX. [Display omitted] •A Bi-Bi sequential steady-state kinetic mechanism for formate oxidase is proposed.•An ionizable group with a pKa value of 4.4 must be protonated for formate binding.•A conformational change occurs before commitment to the first irreversible step.•Formate CH bond cleavage is the rate-limiting step for FOX catalysis.•Both the kcat value and FOX oligomeric state depend on enzyme concentration.