ThnY Is a Ferredoxin Reductase-like Iron-Sulfur Flavoprotein That Has Evolved to Function as a Regulator of Tetralin Biodegradation Gene Expression

• Published in: The Journal of biological chemistry Vol. 286; no. 3; pp. 1709 - 1718
• Main Authors: García, Laura Ledesma, Rivas-Marín, Elena, Floriano, Belén, Bernhardt, Rita, Ewen, Kerstin Maria, Reyes-Ramírez, Francisca, Santero, Eduardo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.01.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Bacteria Biodegradation; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Signal Transduction; Biodegradation, Environmental - drug effects; Cytochromes c - genetics; Cytochromes c - metabolism; Dioxygenases; Electron Transfer; Escherichia coli - enzymology; Escherichia coli - genetics; Evolution, Molecular; FAD; Ferredoxin-NADP Reductase - biosynthesis; Ferredoxin-NADP Reductase - genetics; Ferricyanides - metabolism; Flavoproteins; Flavoproteins - biosynthesis; Flavoproteins - genetics; Gene Expression; Gene Expression Regulation, Bacterial - drug effects; Gene Expression Regulation, Bacterial - physiology; Gene Expression Regulation, Enzymologic - drug effects; Gene Expression Regulation, Enzymologic - physiology; Gene Regulation; NADH; Operon - physiology; Oxidation-Reduction - drug effects; Promoter Regions, Genetic - physiology; Protein Structure, Tertiary; Reductase; Sphingomonas - enzymology; Sphingomonas - genetics; Sphingomonas macrogolitabida; Tetrahydronaphthalenes - metabolism; Tetrahydronaphthalenes - pharmacology; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic - drug effects; Transcription, Genetic - physiology; Gene Regulation; Gene Expression; NADH; Bacteria Biodegradation; Bacterial Signal Transduction; Electron Transfer; FAD; Dioxygenases; Flavoproteins; Reductase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.184648

Previous genetic studies in Sphingomonas macrogolitabida strain TFA have established that expression of genes involved in tetralin biodegradation (thn genes) requires the function of the LysR type activator ThnR and also ThnY. Sequence comparison indicated that ThnY is homologous to bacterial oxygenase-coupled NAD(P)H-dependent ferredoxin reductases. However, ThnY showed substitutions in highly conserved positions of the pyridine nucleotide binding domain of these ferredoxin reductases. ThnY expression is co-regulated with all other genes required for tetralin biodegradation, and presumably thnY is part of the thnCA3A4RY operon. ThnY has been purified, and its biochemical and functional properties were characterized. ThnY was found to be a monomeric orange-brown iron-sulfur flavoprotein (estimated mass of 37,000 Da) containing one non-covalently attached flavin adenine dinucleotide and one plant type ferredoxin 2Fe-2S cluster. It can be efficiently reduced by dithionite, but reduction by pyridine nucleotides was very poor. Consistently, ThnY-dependent reduction of cytochrome c, ferricyanide, or 2,6-dichlorophenolindophenol using NAD(P)H as the electron donor was undetectable or very weak. The addition of ThnY to electrophoretic mobility shift assays containing ThnR and a probe bearing two thn divergent promoters resulted in a 3-fold increase in protein-DNA complex formation affinity, which indicates that ThnY directly promotes thn transcription activation by ThnR.