title: A novel N,N-8-amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces davawensis.
abstract: Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 μm; apparent K(D) for AF = 10.0 μm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.
authors.affiliation: Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Jankowitsch
authors.affiliation: Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Kühm
authors.affiliation: Department of Protein and Cell Science, Merck Serono, Frankfurter Strasse 250, 64271 Darmstadt, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Kellner
authors.affiliation: Center for Biotechnology-CeBiTec, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, Germany.
authors.forename: Jörn
authors.initials: J
authors.lastname: Kalinowski
authors.affiliation: B.R.A.I.N. AG, Darmstädter Strasse 34-36, 64723 Zwingenberg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pelzer
authors.affiliation: Institute of Biochemistry, Graz University of Technology, Petersgasse 12/2, 8010 Graz, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
authors.affiliation: Department of Biotechnology, Institute for Technical Microbiology, Mannheim University of Applied Sciences, 68163 Mannheim, Germany. Electronic address: m.mack@hs-mannheim.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Mack
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008780:Methyltransferases / Q000737:chemistry*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012256:Riboflavin / Q000031:analogs & derivatives / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D008024:Ligands; D011993:Recombinant Fusion Proteins; C009191:roseoflavin; D008780:Methyltransferases; D012256:Riboflavin