The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

Abstract

Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa D-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. ). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.Riboflavin (vitamin B 2 ) analogs have the potential to serve as basic structures for the development of novel anti-infectives (3). Consequently, both the mode of action of riboflavin analogs and the mechanism of resistance to these compounds are of substantial interest. Only very few natural riboflavin analogs are known (24). Moreover, the only known organism to produce a riboflavin analog with antibiotic activity is the grampositive bacterium Streptomyces davawensis (32).S. davawensis synthesizes the anti-vitamin 8-dimethyl-amino-8-demethyl-D-riboflavin, or roseoflavin ( Fig. 1), from riboflavin (27) via 8-amino-and 8-methylamino-8-demethyl-D-riboflavin (22). Roseoflavin is toxic to gram-positive but also to gramnegative bacteria if the compound is able to enter the cell (16). The molecular basis for roseoflavin toxicity is not clear. In all organisms, riboflavin serves as the direct precursor for the cofactors (ribo)flavin-5Ј-monophosphate (FMN) and flavin adenine dinucleotide (FAD) (12). FAD and (to a lesser extent) FMN are active components of flavoproteins, being involved in a wide range of redox and other reactions (15). FMN is synthesized from riboflavin by flavokinase (EC 2.7.1.26), and FAD is produced from FMN by FAD synthetase (EC 2.7.7.2) (2). In microorganisms, both enzymatic activities seem to be present in sufficient amounts, since only traces of free riboflavin are detectable in the cytoplasm (11). Accordingly, cytoplasmic roseoflavin may quickly be converted to the corresponding FMN/ FAD analogs roseoflavin-5Ј-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD). The synthesis of these inactive cofactors may explain the antibiotic activity of roseoflavin ( Fig. 1) (31, 36).S. davawensis is roseoflavin resistant, and the mechanism of self-resistance could involve a flavokinase/FAD synthetase with a high substrate specificity for riboflavin. Such an enzyme would not accept roseoflavin as...

Genome Sequence of the Bacterium Streptomyces davawensis JCM 4913 and Heterologous Production of the Unique Antibiotic Roseoflavin

“…Genes similar to BN159_7986 and BN159_8007 are not present in any of the other Streptomyces genomes (see Table S2 in the supplemental material). Only one gene (BN159_2715) coding for the essential bifunctional flavokinase/FAD synthetase function is present within the genome of S. davawensis, and this enzyme was characterized by us in an earlier work (17). The genes ribBMAH, ribG, and ribA, which were described above to be responsible for riboflavin biosynthesis, are located within the core genome region of S. davawensis.…”

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

“…RoF competes with riboflavin for binding to Lmo1329, which further reduces FMN/FAD levels. In addition to FMN riboswitches and flavin-metabolizing enzymes, flavoproteins are likely targets for flavin analogs, and we propose that one (or several) of the 34 flavoproteins annotated for L. monocytogenes (28) is less active or completely inactive in the presence of either RoFMN or RoFAD as was reported for other bacterial enzymes (5,6,36). The identification of the main flavoprotein target(s) in L. monocytogenes might be rewarding (and is under way) since such a protein might be inhibited by a nonflavin compound and may constitute a completely novel bacterial drug target.…”

“…According to a new nomenclature (35), we propose the name RibCF for the bifunctional flavokinase/FAD synthetase Lmo1329 whereby RibC represents the N-terminal FAD synthetase domain and RibF represents the C-terminal flavokinase domain. Lmo1329 (RibCF) produces AFMN, RoFMN, and AFAD but not RoFAD and thus is different from all other flavokinases/FAD synthetases analyzed so far (5,18,36). Lmo0728 (RibC) is an FAD synthetase and is responsible for the formation of toxic RoFAD in L. monocytogenes.…”

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers

“…Roseoflavin derivatives of FMN and FAD are inactive as coenzymes, apparently due to loss of their oxidizing ability following an intramolecular charge transfer from the 8-dimethylamino group to the pteridine moiety (434). Roseoflavin producers normally convert roseoflavin to analogs of FMN and FAD, so presumably defects in these conversions cannot explain S. davawensis resistance to roseoflavin (155). In roseoflavin-sensitive B. subtilis strains, roseoflavin binds to the FMN riboswitch and thus inhibits transcription of the RF operon (253,335).…”