ThnY is a ferredoxin reductase-like iron-sulfur flavoprotein that has evolved to function as a regulator of tetralin biodegradation gene expression.
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.
Centro Andaluz de Biología del Desarrollo, Consejo Superior de Investigaciones Científicas-Universidad Pablo de Olavide, Carretera de Utrera Km. 1, 41013 Seville, Spain.
Laura Ledesma
LL
García
Elena
E
Rivas-Marín
Belén
B
Floriano
Rita
R
Bernhardt
Kerstin Maria
KM
Ewen
Francisca
F
Reyes-Ramírez
Eduardo
E
Santero
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001673:Biodegradation, Environmental / Q000187:drug effects; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis* / Q000235:genetics; D005292:Ferricyanides / Q000378:metabolism; D005420:Flavoproteins / Q000096:biosynthesis* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects* / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000502:physiology; D009876:Operon / Q000502:physiology; D010084:Oxidation-Reduction / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000502:physiology; D017434:Protein Structure, Tertiary; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics; D013764:Tetrahydronaphthalenes / Q000378:metabolism / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
D001426:Bacterial Proteins; D005292:Ferricyanides; D005420:Flavoproteins; D013764:Tetrahydronaphthalenes; D014157:Transcription Factors; C007931:hexacyanoferrate III; D045304:Cytochromes c; D005287:Ferredoxin-NADP Reductase; C095210:tetralin