title: The flavoenzyme azobenzene reductase AzoR from Escherichia coli binds roseoflavin mononucleotide (RoFMN) with high affinity and is less active in its RoFMN form.
abstract: The Gram-positive bacterium Streptomyces davawensis is the only organism known to produce the antibiotic roseoflavin. Roseoflavin is a structural riboflavin analogue and is converted to the flavin mononucleotide (FMN) analogue roseoflavin mononucleotide (RoFMN) by flavokinase. FMN-dependent homodimeric azobenzene reductase (AzoR) (EC 1.7.1.6) from Escherichia coli was analyzed as a model enzyme. In vivo and in vitro experiments revealed that RoFMN binds to the AzoR apoenzyme with an even higher affinity compared to that of the "natural" cofactor FMN. Structural analysis (at a resolution of 1.07 Å) revealed that RoFMN binding did not affect the overall topology of the enzyme and also did not interfere with dimerization of AzoR. The AzoR-RoFMN holoenzyme complex was found to be less active (30% of AzoR-FMN activity) in a standard assay. We provide evidence that the different physicochemical properties of RoFMN are responsible for its reduced cofactor activity.
authors.affiliation: Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Langer
authors.forename: Shinobu
authors.initials: S
authors.lastname: Nakanishi
authors.forename: Tilo
authors.initials: T
authors.lastname: Mathes
authors.forename: Tanja
authors.initials: T
authors.lastname: Knaus
authors.forename: Alexandra
authors.initials: A
authors.lastname: Binter
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
authors.forename: Tomoko
authors.initials: T
authors.lastname: Mase
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
authors.forename: Matthias
authors.initials: M
authors.lastname: Mack
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D001051:Apoenzymes; D029968:Escherichia coli Proteins; C576819:roseoflavin mononucleotide; D009247:NADH, NADPH Oxidoreductases; C019759:azobenzene reductase; D012256:Riboflavin