Publications

• 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.

  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 methion ... >> More

  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. << Less

  J. Biol. Chem. 286:38275-38285(2011) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural and kinetic studies on RosA, the enzyme catalysing the methylation of 8-demethyl-8-amino-d-riboflavin to the antibiotic roseoflavin.

  Tongsook C., Uhl M.K., Jankowitsch F., Mack M., Gruber K., Macheroux P.

  <h4>Unlabelled</h4>N,N-8-demethyl-8-amino-d-riboflavin dimethyltransferase (RosA) catalyses the final dimethylation of 8-demethyl-8-amino-d-riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present study, we solved the X-ray structure of RosA, and determined th ... >> More

  <h4>Unlabelled</h4>N,N-8-demethyl-8-amino-d-riboflavin dimethyltransferase (RosA) catalyses the final dimethylation of 8-demethyl-8-amino-d-riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present study, we solved the X-ray structure of RosA, and determined the binding properties of substrates and products. Moreover, we used steady-state and rapid reaction kinetic studies to obtain detailed information on the reaction mechanism. The structure of RosA was found to be similar to that of previously described S-adenosylmethionine (SAM)-dependent methyltransferases, featuring two domains: a mainly α-helical 'orthogonal bundle' and a Rossmann-like domain (α/β twisted open sheet). Bioinformatics studies and molecular modelling enabled us to predict the potential SAM and AF binding sites in RosA, suggesting that both substrates, AF and SAM, bind independently to their respective binding pocket. This finding was confirmed by kinetic experiments that demonstrated a random-order 'bi-bi' reaction mechanism. Furthermore, we determined the dissociation constants for substrates and products by either isothermal titration calorimetry or UV/Vis absorption spectroscopy, revealing that both products, RoF and S-adenosylhomocysteine (SAH), bind more tightly to RosA compared with the substrates, AF and SAM. This suggests that RosA may contribute to roseoflavin resistance in S. davawensis. The tighter binding of products is also reflected by the results of inhibition experiments, in which RoF and SAH behave as competitive inhibitors for AF and SAM, respectively. We also showed that formation of a ternary complex of RosA, RoF and SAH (or SAM) leads to drastic spectral changes that are indicative of a hydrophobic environment.<h4>Database</h4>Structural data are available in the Protein Data Bank under accession number 4D7K. << Less

  FEBS J. 283:1531-1549(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.