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.
Highlights
The unusual cofactor F420, a 7,8-didemethyl-8-hydroxy-5deazariboflavin, was originally discovered in various archaea (Cheeseman et al 1972) (Fig. 1)
(WP_011600337.1), RHA1_RS10755 (WP_011595003.1), and RHA1_RS43570 (WP_011600440.1). These genes were amplified from R. jostii RHA1 genomic DNA, cloned into the pET-SUMO vector, and expressed in E. coli C41(DE3) as N-terminal SUMO-hexahistidine-fused proteins using isopropyl β-D-1thiogalactopyranoside (IPTG) as an inducer
This strongly suggests that Rh-FGD1 is a glucose-6-phosphate dehydrogenase
Summary
The unusual cofactor F420, a 7,8-didemethyl-8-hydroxy-5deazariboflavin, was originally discovered in various archaea (Cheeseman et al 1972) (Fig. 1). Due to the unique redox potential (−340 mV) of F420, which is lower than that of FAD (−220 mV) and even of the classical hydrogen carrier NAD(P)+ (Jacobson and Walsh 1984; de Poorter et al 2005), F420H2-dependent enzymes are capable of catalyzing hydrogenation of a wide range of organic compounds which are otherwise recalcitrant to reductive activation such as enones (Taylor et al 2010; Lapalikar et al 2012b; Lapalikar et al 2012a) and imines (Coats et al 1989; Li et al 2009a; Li et al 2009b) in various heterocycles (Schrittwieser et al 2015). Since the identification of the first FGD two decades ago in Daniels’ lab (Purwantini and Daniels 1996), only two FGDs from actinomycetes, namely M. smegmatis and Mtb, have been characterized in detail (Bashiri et al 2007; Bashiri et al 2010) These two FGDs share 37% sequence similarity and belong to an F420-dependent enzyme subgroup within the luciferase-like hydride transferase family. Structural characterization of an FGD from Mtb has been recently described (Bashiri et al 2008)
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