Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase

• DOI: 10.1016/j.jbc.2023.105621
• Publication Date: 2024-01-03T15:19:58Z
• Authors: Prathamesh M. Datar; Soumil Y. Joshi; Sanket A. Deshmukh; E. Neil G. Marsh
• Keywords: Phenazine

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD-family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds are potentially valuable industrial catalysts. We have investigated mechanism PhdA using slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic (DQCA). Detailed analysis pH dependence solvent deuterium isotope effects associated with reaction uncovered unusual kinetic behavior. At low substrate concentrations, substantial inverse effect (SIE) observed Vmax/KM ∼ 0.5 when rates DQCA in H2O D2O compared. Under same conditions, normal SIE 4.15 measured by internal competition for proton transfer product. These apparently contradictory results indicate that values report different steps mechanism. A inventory under Vmax conditions points 'medium effect' as source SIE. Molecular dynamics simulations structure support reduces conformational lability more compact structure, akin active, 'closed' conformer crystal structures some enzymes. Consistent simulations, was found be stable bind tightly, leading rate enhancement conditions.