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

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.DatabaseStructural data are available in the Protein Data Bank under accession number 4D7K.

The Crystal Structure of RosB: Insights into the Reaction Mechanism of the First Member of a Family of Flavodoxin‐like Enzymes

“…This strong binding of AFP by RosB would be compatible with a function as a “chaperone” to guide cytotoxic AFP to the subsequent phosphatase. A similar function was reported for RosA, which also tightly binds its reaction product roseoflavin …”

Metabolic engineering of roseoflavin-overproducing microorganisms

“…Judging from our results, the recombinant strain of S. davaonensis RML7 appears to be a good choice for roseoflavin production since its productivities almost doubled those of its wild-type and more than doubled those of the recombinant C. glutamicum strains (Table 3). Roseoflavin overproduction did not affect growth of S. davaonensis which contains a specialized, RoFMN insensitive FMN riboswitch [26], a roseoflavin exporter [13, 16] and the enzymes RosA and RosB which represent a buffer system for toxic riboflavin analogs [18, 19, 28]. In larger industrial settings, however, C. glutamicum could be a superior host for roseoflavin production as its cultivation is simpler compared to filamentous S. davaonensis [42].…”

“…Moreover, S. davaonensis RosA and RosB tightly bind their toxic reaction products roseoflavin and AFP, preventing their interaction with flavoproteins [18]. A RoFMN-insensitive FMN riboswitch controlling expression of riboflavin genes also shown to contribute to roseoflavin resistance of S. davaonensis [28].…”

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

“…An as yet unknown phosphatase produces AF from AFMN. Finally, the N,N-8-demethyl-8-aminoriboflavin dimethyltransferase RosA (15,16) catalyzes the formation of RoF from AF and S-adenosylmethionine. Inactivation of rosA generated a recombinant S. davawensis strain that produces AF (see Fig.…”