4d7k
From Proteopedia
Crystal structure of N,N-8-amino-8-demethyl-D-riboflavin dimethyltransferase (RosA) from Streptomyces davawensis
Structural highlights
Function[ROSA_STRDJ] Catalyzes the S-adenosyl methionine-dependent conversion of 8-amino-8-demethyl-D-riboflavin (AF) into 8-methylamino-8-demethyl-D-riboflavin (MAF) and roseoflavin (RoF), the last two steps in the biosynthesis of the antibiotic roseoflavin.[1] [2] Publication Abstract from PubMedN,N-8-demethyl-8-amino-D-riboflavin dimethyltransferase (RosA) catalyzes the final dimethylation of 8-demethyl-8-amino-D-riboflavin (AF) to the antibiotic roseoflavin (RoF) in Streptomyces davawensis. In the present study we have solved the X-ray structure of RosA and determined the binding properties of substrates and products. Moreover, we have used steady-state and rapid reaction kinetics to obtain detailed information on the reaction mechanism. The structure of RosA was found to be similar to previously determined S-adenosylmethionine (SAM)-dependent methyltransferases featuring two domains, a mainly alpha-helical "orthogonal bundle" and a Rossmann-like domain (alpha/beta twisted open-sheet). Bioinformatics and molecular modelling enabled us to predict the potential SAM and AF binding sites in RosA suggesting that both substrates, AF and SAM, can bind independently to their respective binding pocket. This was confirmed by kinetic experiments that demonstrated a random-order bi-bi reaction mechanism. Furthermore, we have determined the dissociation constants for substrates and products by either isothermal titration calorimetry or UV-Vis absorption spectroscopy. This revealed that both products, RoF and S-adenosylhomocysteine (SAH), bind more tightly to RosA when compared to 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 inhibition experiments, i.e. RoF and SAH behave as competitive inhibitors for AF and SAM, respectively. We could also show 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. This article is protected by copyright. All rights reserved. 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 MK, Jankowitsch F, Mack M, Gruber K, Macheroux P FEBS J. 2016 Feb 23. doi: 10.1111/febs.13690. PMID:26913589[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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