4D7K

Structural and Kinetic Studies on Rosa, the Enzyme Catalysing the Methylation of 8-Demethyl-8-Amino-D-Riboflavin to the Antibiotic Roseoflavin

(2016) FEBS J 283: 1531

• PubMed: 26913589 Search on PubMedSearch on PubMed Central
• DOI: https://doi.org/10.1111/febs.13690
• Primary Citation of Related Structures:  
  4D7K


• PubMed Abstract: 
  

  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.

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Organizational Affiliation: 

Institute of Biochemistry, Graz University of Technology, Austria.