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

Abstract

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.Tetralin (1,2,3,4-tetrahydronaphthalene) is a bicyclic molecule composed of an aromatic and an alicyclic moiety, which is found at low concentrations in different crude oils, and it is also industrially produced for its use as an organic solvent. Biodegradation of tetralin has been characterized most extensively in Sphingomonas macrogolitabida strain TFA. The genes required for tetralin biodegradation (thn genes) have been sequenced, and the functions of the encoded proteins have been characterized to elucidate the biodegradation pathway (1-4) that yields pyruvate and pimelate, which is subsequently metabolized via ␤-oxidation (5). Rieske type oxygenases are multicomponent enzymes known to initiate the oxidative biodegradation of various environmentally hazardous aromatic compounds in bacteria, such as crude oil components, polycyclic aromatic hydrocarbons, and heterocyclic aromatic compounds, in a reaction requiring oxygen and an external electron supply (6). In this reaction, the electrons are transferred from NAD(P)H via flavin and [2Fe-2S] redox centers to the terminal oxygenase component that has the catalytic active site. Tetralin dioxygenase is encoded by the thnA1 and thnA2 genes, whereas the ferredoxin and the ferredoxin reductase components of the electron transfer system are encoded by thnA3 and thnA4, respectively. According to the number of components and the nature of the redox centers of the electron transport chain, bacterial oxygenases have been classified into three main classes, classes I, II, and III (7-9). The tetralin dioxygenase s...

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A novel NADPH-dependent reductase of Sulfobacillus acidophilus TPY phenol hydroxylase: expression, characterization, and functional analysis

“…The natural substitution of this conserved Cys residue observed in MhpP is rare in FNR and FNR-like proteins. However, natural loss of the highly conserved Cys of yXCGp was reported for an iron-sulfur flavoprotein involved in tetralin biodegradation (García et al 2011). Substrate specificity analyses confirmed that MhpP preferentially utilizes NADPH rather that NADH as an electron donor (Table 3).…”

Biochemical Characterization of Inducible ‘Reductase’ Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4

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