Kinetic Characterization of F420-Dependent Sugar-6-Phosphate Dehydrogenase from Cryptosporangium arvum

Abstract

F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD) catalyzes the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone (6PG). Recent phylogenetic analyses have identified a new subclass of these enzymes, F₄₂₀-dependent sugar-6-phosphate dehydrogenases (FSDs), which act on a broader range of 6-phosphate sugars, including fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P). One such enzyme from Cryptosporangium arvum (Cryar-FGD) was characterized by using binding assays and kinetic analyses, nuclear magnetic resonance (NMR), and mass spectrometry. Results showed strong binding affinities for all substrates. Steady-state kinetic analysis revealed that G6P has the highest catalytic efficiency, with a k_cat(app) of 6.4 ± 0.2 s^–1, compared to 1.4 ± 0.1 s^–1 for F6P and 0.32 ± 0.02 s^–1 for M6P. Pre steady-state spectral features for the G6P reaction resembled those of Mycobacterium tuberculosis FGD. While the F6P reaction displayed distinct spectral features, F₄₂₀ reduction was still observed. In contrast, the spectra for the M6P reaction were markedly different from those of G6P and F6P. Across all substrates, no catalytic intermediates were detected, and hydride transfer was not rate-limiting. As with G6P, the reaction with F6P also produced 6PG. Notably, NMR data showed that F6P was isomerized to G6P, suggesting isomerase activity. In contrast, M6P induced only spectral shifts with no evidence of isomerization or 6PG formation. However, mass spectrometry confirmed oxidized products for all three sugars, each with a mass of 299.0 ± 0.1. Collectively, these findings reveal that Cryar-FGD exhibits both dehydrogenase and isomerase activity, uncovering a newly identified dual enzymatic function and establishing its role as a multifunctional enzyme.