title: 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 F420cofactor 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.
authors.affiliation: Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.|Scuola Universitaria Superiore IUSS Pavia, Piazza della Vittoria 15, 27100, Pavia, Italy.|Faculty of Pharmacy, University of Medicine and Pharmacy, Ho Chi Minh City, 41 Dinh Tien Hoang Street, Ben Nghe Ward, District 1, Ho Chi Minh City, Vietnam.
authors.forename: Quoc-Thai
authors.initials: QT
authors.lastname: Nguyen
authors.affiliation: Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.|Membrane Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Trinco
authors.affiliation: Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Binda
authors.affiliation: Department of Biology and Biotechnology, University of Pavia, Via Ferrata 1, 27100, Pavia, Italy. andrea.mattevi@unipv.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mattevi
authors.affiliation: Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands. m.w.fraaije@rug.nl.
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D001673:Biodegradation, Environmental; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005415:Flavins / Q000378:metabolism*; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010088:Oxidoreductases / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012240:Rhodococcus / Q000201:enzymology*; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005415:Flavins; D011994:Recombinant Proteins; C019319:5-deazaflavin; D019298:Glucose-6-Phosphate; C015084:coenzyme F420; D010088:Oxidoreductases; D005954:Glucosephosphate Dehydrogenase; D012256:Riboflavin
keywords: Deazaflavoenzymes; F420; Glucose-6-phosphate dehydrogenase; Rhodococcus