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.

Cofactor F420-Dependent Enzymes: An Under-Explored Resource for Asymmetric Redox Biocatalysis

“…IS2 contains two antiparallel β-strands, and IS3 contains a helical bundle at the C-terminus of the β-barrel and contains the remainder of the substrate-binding pocket ( Figure 3). All structures solved to date from the LLHT family contain a non-prolyl cis peptide in β3 [23][24][25][26]. Recent phylogenetic reconstructions have shown that the F 420 -dependent LLHTs form two clades: the F 420 -dependent reductases and the F 420 -depented dehydrogenases [27].…”

“…The second contains the F 420 -dependent glucose-6-phosphate dehydrogenases (FGDs) from Mycobacteria and Rhodococcus, while the third appear to be more general sugar-phosphate dehydrogenases [27]. In contrast to the heterodimeric structure of bacterial luciferase, the F 420 -dependent dehydrogenases form homodimers with the dimer interface burying a relatively large portion of the surface area of the monomers (≈2000 Å 2 , roughly 15% Catalysts 2019, 9, 868 5 of 18 of the total surface area) [24][25][26]. A number of enzymes involved in the F 420 -dependent degradation of nitroaromatic explosives, such as picrate and 2,4-dinitroanisole, appear to belong to the LLHT family as well [32,33].…”

“…Catalysts 2019, 9, x FOR PEER REVIEW 5 of 19 Å 2 , roughly 15% of the total surface area) [24][25][26]. A number of enzymes involved in the F420dependent degradation of nitroaromatic explosives, such as picrate and 2,4-dinitroanisole, appear to belong to the LLHT family as well [32,33].…”

“…The LLHT family contains several enzymes with alcohol oxidase or ketoreductase activity (Table 1; Figure 4). The F 420 -dependent glucose-6-phosphate dehydrogenases of several species have been investigated [25,26,39]. Although an extensive survey of their substrate ranges has yet to be conducted, it has been demonstrated that glucose is a substrate for the Rhodococcus jostii RHA1 enzymes [26].…”

“…The F 420 -dependent glucose-6-phosphate dehydrogenases of several species have been investigated [25,26,39]. Although an extensive survey of their substrate ranges has yet to be conducted, it has been demonstrated that glucose is a substrate for the Rhodococcus jostii RHA1 enzymes [26]. An F 420 -dependent alcohol dehydrogenase (ADH) from Methanogenium liminatans has been shown to catalyze the oxidation of the short chain aliphatic alcohols 2-propanol, 2-butanol and 2-pentanol (85, 49 and 23.1 s −1 k cat , 2.2, 1.2 and 7.2 mM K M respectively) [40], but it was unable to oxidize primary alcohols, polyols or secondary alcohols with more than five carbons.…”