title: Anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii possesses both FMN and FAD in two distinct and independent active sites.
abstract: Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bergner
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, A-8010 Graz, Austria.
authors.forename: Tea
authors.initials: T
authors.lastname: Pavkov-Keller
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Kreuzer
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Jakob
authors.initials: J
authors.lastname: Kowaliuk
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Plank
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Runggatscher
authors.affiliation: Institute of Chemistry, University of Graz, A-8010 Graz, Austria.
authors.forename: Nikolaus G
authors.initials: NG
authors.lastname: Turrini
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Zucol
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria.
authors.forename: Silvia
authors.initials: S
authors.lastname: Wallner
authors.affiliation: Institute of Chemistry, University of Graz, A-8010 Graz, Austria.
authors.forename: Kurt
authors.initials: K
authors.lastname: Faber
authors.affiliation: Institute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria.
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.affiliation: Institute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria. Electronic address: peter.macheroux@tugraz.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
mesh_terms: D020627:Azoarcus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003065:Coenzyme A / Q000737:chemistry; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; D003065:Coenzyme A
keywords: Flavin; Microcalorimetry; Monooxygenase; Old Yellow Enzyme; Reductase; Small-angle X-ray scattering