A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis

Jankowitsch F, Kuehm C, Kellner R, Kalinowski J, Pelzer S, Macheroux P, Mack M (2011)
Journal of Biological Chemistry 286(44): 38275-38285.

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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 mu M; apparent K(D) for AF = 10.0 mu M; k(cat) = 0.37 +/- 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.
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Jankowitsch F, Kuehm C, Kellner R, et al. A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis. Journal of Biological Chemistry. 2011;286(44):38275-38285.
Jankowitsch, F., Kuehm, C., Kellner, R., Kalinowski, J., Pelzer, S., Macheroux, P., & Mack, M. (2011). A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis. Journal of Biological Chemistry, 286(44), 38275-38285.
Jankowitsch, F., Kuehm, C., Kellner, R., Kalinowski, J., Pelzer, S., Macheroux, P., and Mack, M. (2011). A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis. Journal of Biological Chemistry 286, 38275-38285.
Jankowitsch, F., et al., 2011. A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis. Journal of Biological Chemistry, 286(44), p 38275-38285.
F. Jankowitsch, et al., “A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis”, Journal of Biological Chemistry, vol. 286, 2011, pp. 38275-38285.
Jankowitsch, F., Kuehm, C., Kellner, R., Kalinowski, J., Pelzer, S., Macheroux, P., Mack, M.: A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis. Journal of Biological Chemistry. 286, 38275-38285 (2011).
Jankowitsch, Frank, Kuehm, Christian, Kellner, Roland, Kalinowski, Jörn, Pelzer, Stefan, Macheroux, Peter, and Mack, Matthias. “A Novel N,N-8-Amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) Catalyzing the Two Terminal Steps of Roseoflavin Biosynthesis in Streptomyces davawensis”. Journal of Biological Chemistry 286.44 (2011): 38275-38285.
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Langer S, Hashimoto M, Hobl B, Mathes T, Mack M., J. Bacteriol. 195(18), 2013
PMID: 23836860
Genome sequence of the bacterium Streptomyces davawensis JCM 4913 and heterologous production of the unique antibiotic roseoflavin.
Jankowitsch F, Schwarz J, Ruckert C, Gust B, Szczepanowski R, Blom J, Pelzer S, Kalinowski J, Mack M., J. Bacteriol. 194(24), 2012
PMID: 23043000
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Pedrolli DB, Matern A, Wang J, Ester M, Siedler K, Breaker R, Mack M., Nucleic Acids Res. 40(17), 2012
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