Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1

Abstract

Cofactor F₄₂₀, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F₄₂₀-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F₄₂₀-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F₄₂₀ + glucose-6-phosphate → F₄₂₀H₂ + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F₄₂₀ cofactor required for numerous F₄₂₀H₂-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F₄₂₀-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F₄₂₀-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F₄₂₀H₂-dependent reductases in industry and bioremediation.

Keywords: Deazaflavoenzymes; F420; Glucose-6-phosphate dehydrogenase; Rhodococcus.

Fig. 1

Reaction catalyzed by F₄₂₀-dependent dehydrogenase (FGD). Glucose-6-phosphate is oxidized into 6-phosphogluconolactone by FGD concomitantly with the formation of the reduced F₄₂₀ coenzyme, which is subsequently employed by various F₄₂₀H₂-dependent reductases

Fig. 2

Effect of pH on Rh-FGD1 activity. The reaction contains 40 mm Britton–Robinson buffer, 100 nm Rh-FGD1, 20 μM F₄₂₀, and 1.0 mm G6P, and activity was monitored by following the absorbance at 401 nm (isosbestic point of F₄₂₀) for 300 s at 25 °C

Fig. 3

Melting temperatures of Rh-FGD1 in different buffer (a) and additive (b) conditions measured by the Thermofluor® technique. Buffers were used at a concentration of 100 mm unless otherwise indicated. The error bars represent SD from the three replicates. Buffer A succinic acid/ NaH₂PO₄/ glycine = (2:7:7). Buffer B citric acid/ CHES/ HEPES = (2:4:3). Ac acetate, Am ammonium, DTT dithiothreitol

Fig. 4

Two-substrate kinetic analysis for Rh-FGD1 via double reciprocal plots of reaction rates against a G6P or b F₄₂₀ concentrations. These lines intercept at one point, corresponding to the formation of a ternary complex Rh-FGD1:G6P:F₄₂₀ to generate 6-phosphogluconolactone and F₄₂₀H₂

Fig. 5

Crystal structure of Rh-FGD1 from Rhodococcus jostii RHA1. a Ribbon diagram of the Rh-FGD1 dimer showing the (α/β)₈ TIM-barrel architecture of the two monomers colored in light blue (monomer A) and green (monomer B), respectively. The disordered region in each monomer is represented by a dashed line corresponding to residues 254–263 and 250–279 in monomers A and B, respectively. b Superposition of the Rh-FGD1 dimer (colored as in a) onto the homologous Mtb-FGD1 [in white, 84% sequence identity, PDB ID 3Y4B (Bashiri et al. 2008)] with its F₄₂₀ cofactor bound (carbon, oxygen, nitrogen, and phosphorus atoms in white, red, blue and magenta, respectively). c The nonprolyl cis peptide bond (connecting Ser72 and Val73) and Met74 in a double conformation (sulfur atoms in green) are fitted to the initial 2F _o − F _c electron density map contoured at 1.2 σ (brown chicken-wire). As a reference, the cofactor F₄₂₀ from the Mtb-FGD1 structure (superposed as in b) is drawn with shaded colors. d Close-up of the Rh-FGD1 active site superposed to Mtb-FGD1 as in b. The Mtb-FGD1 inhibitor citrate (carbon in gray) is shown bound to the active site. Putative residues involved in substrate binding are labeled with the corresponding Mtb-FGD1 residues in parentheses. The δ, ϵ carbon and ζ nitrogen atoms of K197, and the guanidinium group of R282 side chains were not visible in the electron density and were not included in the final model

Fig. 6

Comparison between the active site of Rh-FGD1 (blue) with that of Mtb-FGD1 (3B4Y, white), Adf (1RHC, coral), and Mer (1Z69, green). For clarity, only the F₄₂₀ from Mtb-FGD1 is shown. The insertion regions of Mtb-FGD1, Adf, and Mer corresponding to the highly disordered segment in Rh-FGD1 (residues 254–263, represented by a dashed line) are highlighted in bold style. The orientation of the molecule is approximately 90° rotated along an axis perpendicular to the plane of the paper with respect to that in Fig. 5c. Color coding for atoms is as in Fig. 5b