The Group of Matthias Mack at Mannheim University of Applied Sciences

Why do we study vitamin analogs such as the antibiotic roseoflavin from the bacterium Streptoymces davawensis?

• Vitamin analogs have a good potential to serve as basic structures for the development of novel antiinfectives and will help to replenish the arsenal of antimicrobials urgently needed to fight multiresistant bacterial pathogens.
• Many microorganisms have efficient vitamin transporters, which catalyze the uptake of vitamins but also vitamin analogs. Thus, the delivery of the antivitamin to the target molecules within the target cells is ensured (we call it the “Trojan Horse” principle) (Fig. 6).
• Vitamin analogs in principle have multiple cellular targets, since many vitamins (as precursors of enzyme cofactors), are active at more than one site in the cell. As a result, the frequency of developing resistance to antimicrobials based on vitamin analogs is expected to be significantly lower (Fig. 6).
• Some vitamins already are synthesized on an industrial scale using microorganisms. Thus, the development of a production process for vitamin analogs is straightforward.
• Vitamin analogs are especially interesting for pharmaceutical applications since these compounds have coevolved in close contact to potential target cells. On the one hand natural antimetabolites have been “optimized” throughout evolution with respect to their antibiotic function. On the other hand they may in general have a lower toxicological potential, since they have coevolved with cellular structures of the producer.

 Where are vitamin analogs found in nature?

Only three naturally occurring vitamin analogs with an antibiotic function have been featured in publications: the vitamin B₁ analog bacimethrin, the vitamin B₆ analog ginkgotoxin and the vitamin B₂ (riboflavin) analog roseoflavin. Possibly, flavin analogs with antibiotic activity are more widespread than anticipated. The same could be true for flavin analogs yet to be discovered, which could constitute tools for cellular chemistry allowing a further extension of the catalytic spectrum of flavoenzymes.

What did we find out until now?

• Biosynthesis of roseoflavin:

  Roseoflavin can be considered a natural antimetabolite and was postulated to be biosynthesized from riboflavin via the key intermediate 8-demethyl-8-amino-riboflavin (AF). The required site-specific substitution of one of the methyl groups on the dimethylbenzene ring of riboflavin by an amino group (to give AF) is challenging. We found that the enzyme specified by the gene BN159_7989 (RosB) from S. davawensis is able to carry out a whole set of chemical reactions: starting with riboflavin-5’-phosphate, the final product of BN159_7989 is 8-demethyl-8-amino-riboflavin-5’-phosphate (AFP). The conversion of AF to RoF is carried out by the N,N-8-amino-8-demethyl-riboflavin dimethyltransferase RosA. The phosphatase generating AF from AFP is still unknown. In contrast, the enzyme which produces riboflavin-5’-phosphate from riboflavin and ATP is known, we named it RibC. Thus, aside from the dephosphorylation step, roseoflavin biosynthesis has been elucidated (Fig. 7).

• Biosynthesis of riboflavin

  Riboflavin is the direct precursor to roseoflavin and we therefore study riboflavin biosynthesis in S. davawensis and also in other bacteria. The highlight of this research was the identification of a novel regulator RibR in the bacterium Bacillus subtilis which interacts with the FMN riboswitch RNA and modulates riboflavin biosynthesis.

• Roseoflavin: Molecular mechanism of action, flavoproteins as targets

  Riboflavin-5’-phosphate (flavin mononucleotide or FMN) and flavin adenine dinucleotide (FAD) are cofactors of flavoproteins/flavoenzymes, which have a wide variety of different biological functions. The number of flavin-dependent proteins varies greatly in different organisms (and amongst pathogens) and covers a range from approximately 0.1% to 3.5% of the proteome. Following import flavins are metabolised by bacterial flavokinases/FAD synthetases. These enzymes generate FMN (from riboflavin and ATP) and FAD (from FMN and ATP). These enzymes also are responsible for the formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide (RoFAD). Flavoproteins were found to be less active or completely inactive when RoFMN or RoFAD were bound instead (Fig. 8).

• Roseoflavin: Molecular mechanism of action, riboswitches as target

  Riboswitches represent gene regulatory systems that consist of a metabolite-responsive aptamer and an overlapping expression platform and are predominantly located in the 5’-untranslated regions (UTRs) of mRNAs. FMN riboswitches regulate riboflavin biosynthesis and transport are turned “off” in the presence of RoFMN causing riboflavin deficiency and represent additional targets for RoF (see Fig. 6). Roseoflavin is the only natural antimetabolite known to affect riboswitches which especially aroused our interest. Our work on the mechanism of action of RoF shows (for the first time) that RoF indeed has a physiological consequence for RoF treated cells. The activity of the enzyme which is responsible for the synthesis of riboflavin (RibB), is strongly reduced in RoF treated cells which results in reduced riboflavin levels (Fig. 9). The reduced riboflavin levels in turn lead to reduced growth. Similar data were generated for E. coli and L. monocytogenes. We found that these cells show reduced cellular FMN/FAD levels which probably leads to less active flavoenzymes.

• Roseoflavin

  The molecular mechanism of resistance: The roseoflavin producer S. davawensis is roseoflavin resistant, the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and (at the same time) to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that one of the FMN riboswitches in S. davawensis, the ribB FMN riboswitch, is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands. This appears to be the main mechanism of resistance although we are pretty sure that a roseoflavin exporter supports S. davawensis with regard to roseoflavin resistance.