The Group of Matthias Mack at Mannheim University of Applied Sciences

What kind of research do we do?

The Gram-positive soil bacteria Streptomyces davawensis (Fig. 1) and Streptomyces cinnabarinus (Fig. 2) are the only organisms known to produce the vitamin analog roseoflavin. We study the biosynthesis of roseoflavin, its molecular mechanism of action and the resistance mechanism of the producer cells in order to pave the way for the structured analysis of other vitamin analogs yet to be discovered.

Notably, roseoflavin is active against a variety of pathogens e.g. Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, Plasmodium falciparum and others.

The following pictures and the text show what we know about roseoflavin (until now) and why we are interested in this unique compound. Please check our publications which of course provide you with more detailed information with regard to roseoflavin. These reviews are a starting point.

Pedrolli DB, Jankowitsch F, Schwarz J, Langer S, Nakanishi S, Frei E, Mack M. 2013. Riboflavin analogs as antiinfectives: occurrence, mode of action, metabolism and resistance. Curr Pharm Des, 19(14):2552-60.

Pedrolli DB, Mack M. 2014. Bacterial flavin mononucleotide riboswitches as targets for flavin analogs. Methods Mol Biol, 1103:165-76

The most important problem with regard to roseoflavin, however, has not yet been solved. What is the function of this antibiotic in its natural habitat (the soil)? If you are interested in contributing to solve this problem, please get in contact with Matthias Mack.

Fig. 1 Colonies of the bacterium Streptomyces davawensis. S. davawensis was grown on a solid medium containing starch and yeast extract. S. davawensis was first isolated by Otani and coworkers near the airport of Davao City (Philippines). The red color is due to the secretion of (red) roseoflavin by S. davawensis.

Fig. 3 The bacteria Streptomyces davawensis and Streptomyces cinnabarinus apparently are closely related. The small pictures show electron microscopic images of exospores formed by the two bacteria.

Fig. 2 Colonies of the bacterium Streptomyces cinnabarinus. S. cinnabarinus was grown on yeast malt extract agar. S. cinnabarinus was isolated from a soil sample (cinnabar = red). The red color is due to the secretion of (red) roseoflavin by S. cinnabarinus.

Fig. 4 The linear chromosome of Streptomyces davawensis has been sequenced and analysed in collaboration with CeBiTec (U Bielefeld, Kalinowski group).

Fig. 5 The antibiotic roseoflavin is synthesized by the bacterium Streptomyces davawensis grown on a nutrient broth containing yeast extract and starch.

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.

Fig. 6 Molecular targets for the antibiotic roseoflavin from Streptomyces davawensis or “why does roseoflavin inhibit growth of a variety of pathogens”. The structural riboflavin analog roseoflavin is taken up by riboflavin transporters (these transporters are present in many organisms). Roseoflavin is converted to the flavin cofactor analogs RoFMN (FMN analog) and RoFAD (FAD analog) by ATP-dependent cellular flavokinases/FAD synthetases. These enzymes are present in all organisms. RoFMN and RoFAD combine with flavoenzymes which are less active or inactive when containing RoFMN or RoFAD instead of FMN or FAD. In addition, RoFMN blocks FMN riboswitches, genetic elements, which regulate riboflavin biosynthesis and transport in many microorganisms. The result of this inactivation of FMN riboswitches is riboflavin auxotrophy i.e. cells do not have sufficient amounts of the vitamin riboflavin. Roseoflavin may have up to 80 target structures in a cell!

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).

Fig. 7 Roseoflavin (RoF) biosynthesis in Streptomyces davawensis. A vitamin is converted to an antibiotic - the path from riboflavin (vitamin B2) to the antibiotic roseoflavin (RoF) is shown. The partial reactions from riboflavin-5’-phosphate (RP or flavin mononucleotide, FMN, compound 2) to the intermediate 8-demethyl-8-amino-riboflavin-5’-phosphate (AFP, compound 5) were unknown and hypothesized to require a series of enzymes. We now show that this complex reaction is catalyzed by a single enzyme BN159_7989 or RosB. RP is formed from riboflavin and ATP by the flavokinase RibC.[8b] The first RosB reaction, the conversion of RP to OHC-RP (CP1), occurs in the presence of oxygen only. Other substrates or cofactors are not required. The conversion of OHC-RP to HO2C-RP depends on thiamine, oxygen is not necessary. HO2C-RP is decarboxylated and transaminated to AFP in the presence of thiamine and glutamate with the concomitant synthesis of 2-oxoglutarate (2-OG). This reaction occurred also in the absence of oxygen. The addition of NAD+ (but not of NADP+) stimulated RosB catalysed AFP formation by a factor of 1.7, however, the reaction occurred also without the addition of NAD+. The S-adenosyl methionine (SAM) dependent dimethyltransferase RosA is responsible for the conversion of AF to RoF (via MAF). As a side-product S-adenosylhomocysteine (AHC) is formed. Notably, AFP is not a substrate for RosA, the dephosphorylation of AFP is carried out by a yet unknown enzyme.

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).

Fig. 8: RoFMN and/or RoFAD are ligands for flavoproteins of Escherichia coli. Different E. coli strains overproducing 40 different (putative) E. coli (flavo)proteins were grown in the presence of limiting amounts of riboflavin. IPTG was added to the cultures in order to induce oversynthesis of the different recombinant His6-tagged E. coli flavoproteins. At the same time roseoflavin was added and the strains were grown to the stationary phase. The recombinant proteins were purified by affinity chromatography and analyzed with regard to their flavin cofactor content using HPLC/MS. The flavin content (%) shows how much RoFMN/RoFAD (black columns) and/or FMN/FAD (grey columns) was found in flavoproteins purified from roseoflavin treated strains. Almost all flavoproteins (besides Lpd) bind RoFMN/RoFAD and most likely are inactivated by these cofactor analogs.

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.

Fig. 9 RoFMN binding in the active site of the enzyme AzoR from Escherichia coli. The structure was solved by analyzing the red RoFMN containing AzoR crystal. The cofactor analog RoFMN reduces AzoR activity by 70%, the reason is the drastically reduced reduction potential of RoFMN (when compared to FMN). Surprisingly, RoFMN binds even better to the active site of AzoR when compared to FMN. This can be explained by additional hydrophobic contacts to amino acids of the active site which are possible through the dimethylamino group of RoFMN.

Fig. 10 Expression of the genes ribBMAH responsible for riboflavin biosynthesis in Streptomyces davawensis and in Streptomyces coelicolor are controlled by an FMN riboswitch. The gene ribB encodes riboflavin synthase, which catalyzes the last step in riboflavin biosynthesis. S. coelicolor RibB activity drops when cells are treated with roseoflavin. In contrast, RibB activity in S. davawensis is not affected by roseoflavin. The FMN riboswitch of S. davawensis is special, it is not turned “off” by roseoflavin.

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.

Fig. 11 (a) Sequence, secondary structure, and expected transcriptional intermediates of the 5’-UTR of the ribB mRNA from S. davawensis (RBS, ribosomal binding site, nucleotides in red). The key nucleotide pair (61AC75) with respect to RoFMN resistance is boxed. Nucleotides differing to the S. coelicolor aptamer are boxed yellow. Nucleotides marked brown are responsible for anti-sequestration. (b) Nucleotide sequences of aptamer portions of FMN riboswitches from different Streptomycetes. The aptamers are highly similar and only the nucleotides differing to the S. davawensis ribB FMN riboswitch (top sequence) are shown. The S. davawensis aptamer is the only sequence containing an A at position 61 (boxed). The other Streptomyces species show either a G or a T (U) at this position.

We gratefully acknowledge funding by …

The Federal Ministry of Education and Research (BMBF)
The Deutsche Forschungsgemeinschaft (DFG, German Research Foundation
The Albert und Anneliese Konanz-Stiftung
The Landesstiftung Baden-Württemberg
The State of Baden-Württemberg

We thank the The Hartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS) and the Karlsruhe Institute of Technology (KIT) for cooperation with regard to joint PhD programs.

We thank our cooperation partners …

Prof. Dr. Peter Macheroux, TU Graz, Austria (reaction mechanisms of enzymes)
Prof. Dr. Danielle Pedrolli, UNESP, Brazil (riboswitches)
Prof. Dr. Beatrix Süss, TU Darmstadt (riboswitches)
Dr. Joachim Wink, Helmholtz Centre for Infection Research (HZI), Braunschweig (biology of Streptomycetes)
Dr. Ulrich Ermler, Max Planck Institut für Biophysik, Frankfurt (structural biology)
PD Dr. Jürgen Stolz, TU München (transport of flavins)
PD Dr. Bertolt Gust, U Tübingen (genetic tools for Streptomycetes)
Prof. Dr. Peter Graumann, U Marburg (fluorescence microscopy)
Prof. Dr. Reinhard Fischer, KIT Karlsruhe (molecular biology of fungi)
Dr. Tilo Mathes, HU Berlin (biophysical characterization of flavoenzymes)
Dr. Masayuki Hashimoto, Tainan University, Taiwan (gene deletion in Escherichia coli)
Prof. Dr. Jonas Contiero, UNESP Rio Claro, Brasil (biofuels)
Dr. Kevin Saliba, Australian National University, Australien (molecular mode of action of roseoflavin on Plasmodium falciparum)
Prof. Dr. Jörn Kalinowski, CeBiTec, U Bielefeld (genomics and transcriptomics)
Prof. Dr. Uwe Sauer ETH Zürich (metabolomics)
Dr. Roland Kellner, Merck KGaA (identification of proteins)
Prof. Dr. Masaru Tanokura, U Tokio, Japan (structural biology)
Prof. Dr. Tadgh Begley, U Texas, USA (synthesis of flavin analogs)