Vitamin Analogs As Antiinfectives: Occurrence, Mode of Action, Metabolism and Production

Vitamin analogs as anti-infectives: Occurrence, mode of action,
metabolism and production

Danielle Biscaro Pedrolli, Frank Jankowitsch, Simone Langer,
Julia Schwarz and Matthias Mack

Institute for Technical Microbiology,
Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10, 68163 Mannheim,
Germany.

Antimetabolites are
compounds, which are structurally similar to molecules needed to carry out
primary metabolic reactions. The inhibitory activity of an antimetabolite
depends on its successful competition with the natural substrate, ligand,
modulator or cofactor of a given biomolecule. Antimetabolites are indispensable
as molecular tools in order to understand biological processes. Beyond that, antimetabolites
are used as anti-infectives and anti-cancer drugs and
also are employed as preserving agents, pesticides or insecticides.

Natural vitamin analogs with antibiotic
function can be considered as natural antimetabolites and aroused our interest
for the following reasons:

1. From an evolutionary standpoint the ?development?
of vitamin analogs appears to be economical since their precursor molecules, vitamins,
are already available in producer cells and merely modifying enzymes are needed
to convert the vitamin into an antibiotic.

2. Many microorganisms (target organisms) have
efficient vitamin transporters. These transporters also catalyze the uptake of
vitamin analogs and ensure the delivery of the antibiotic to the target
molecules in a cell.

3. Since most vitamins are active at more
than one site, vitamin analogs in principle have multiple cellular targets and the
frequency of appearance of resistant strains is expected to be lower.

4. Many vitamins are synthesized on an
industrial scale using microorganisms. Once the biosynthetic pathway for a
certain vitamin analog is known, an already established vitamin bioprocess can
be used as a starting point for a vitamin analog bioprocess.

5. Natural antimetabolites are thought to
in general have a lower toxicological potential since these compounds have
coevolved in close contact to cellular structures.

Three examples of naturally occurring
vitamin analogs with antibiotic/toxin function have been featured in
publications: Bacimethrin, ginkgotoxin and roseoflavin. Bacimethrin is a
thiamine (vitamin B1) analog produced by Bacillus megaterium and Streptomyces
albus. Ginkgotoxin is a neurotoxin occurring in Ginkgo biloba and is
structurally related to vitamin B6. Roseoflavin is a riboflavin (vitamin B2)
analog produced by the Gram-positive bacteria Streptomyces
davawensis and Streptomyces cinnabarinus (1). Roseoflavin is studied in our laboratory as a model compound. We investigate
the biosynthesis of roseoflavin,
its possible large-scale production, its metabolization, its mode of action and
the resistance mechanism of the producer organisms in order to pave the way for
the structured analysis of other vitamin analogs to be discovered. It was
postulated that roseoflavin is synthesized from GTP and ribulose-5-phosphate
through riboflavin, 8-amino-riboflavin and 8-methylamino-riboflavin (2). Recently, the first enzyme of the
roseoflavin biosynthetic pathway has been identified
in S. davawensis, an
(S)-adenosyl methionine-dependent N,N-8-amino-riboflavin dimethyltransferase (EC
2.1.1.93), which converts 8-amino-riboflavin in two steps to roseoflavin
(3). The corresponding gene rosA was found to be located in a
cluster comprising a total of 10 genes. The remaining genes of this cluster were
shown to not be involved in roseoflavin biosynthesis. Employing cluster
deletion and heterologous expression experiments a second gene cluster was identified
which directs the synthesis of 8-amino-riboflavin. The genes/enzymes of this cluster are currently analyzed with
regard to their function, which eventually should lead
to the elucidation of the complete roseoflavin biosynthetic pathway. About eight thousand
tons of riboflavin are produced annually and cost-effective biotechnological
riboflavin production processes were developed using e.g. Bacillus subtilis
and Ashbya gossypii. We anticipate that an economic bio-production process
can also be established for roseoflavin, once its biosynthetic pathway is fully
understood.

The mechanism of action of roseoflavin was
studied in considerable detail in our laboratory (1). For a variety of microorganisms it was shown that roseoflavin is
an excellent substrate for riboflavin transporters (4). Moreover, it was found that roseoflavin quickly and almost
quantitatively is converted to the flavin cofactor analogs roseoflavin
mononucleotide (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) by
flavokinases (EC 2.7.1.26) and FAD synthetases (EC 2.7.7.2) (5,6). These enzymes are present in all organisms.
Thus, RoFMN and RoFAD (rather than roseoflavin) are the effector molecules in
target cells. RoFMN was reported to reduce expression of genes involved in
riboflavin biosynthesis and/or transport in B. subtilis, Streptomyces
coelicolor and the human pathogen Listeria monocytogenes (7,8). These genes are all controlled by FMN riboswitches, regulatory genetic
elements, which are negatively affected by RoFMN. This reduction of gene
expression explains at least in part why roseoflavin acts as an antibiotic. For
example, reduced expression of the FMN riboswitch controlled riboflavin
biosynthetic genes ribEMAH in S. coelicolor (caused by RoFMN) led
to a significantly decreased level of riboflavin synthase (RibE, EC 2.5.1.9) activity
and consequently to reduced supply of riboflavin (9). The addition of roseoflavin to riboflavin auxotrophic L.
monocytogenes resulted in reduced expression of the FMN riboswitch
controlled riboflavin transporter gene lmo1945 and to reduced supply
with riboflavin as well (7). However, RoFMN and RoFAD were found to be also active at other
sites in target cells. Approximately 1-3% of all bacterial proteins depend on
the riboflavin derived cofactors FMN or FAD (10) and thus naturally constitute additional targets for RoFMN and RoFAD.
Indeed, some of these FMN- or FAD-dependent proteins (?flavoproteins?) were found to be less active or completely
inactive in combination with RoFMN or RoFAD (6). Moreover, 37 out of 38 Escherichia coli flavoproteins were
shown to contain either RoFMN or RoFAD when cells were treated with roseoflavin
(11) indicating that roseoflavin indeed has multiple targets and has the
potential to exert a broad negative effect on cellular physiology (Fig. 1). No FMN riboswitches or similar
flavin-binding control elements were found in humans or any other mammal.
However, since human flavokinase and FAD synthetase convert roseoflavin to RoFMN
and RoFAD it is possible that flavin analogs (and/or degradation products of
flavin analogs) negatively interfere with human metabolism ( none">12).

S. davawensis is naturally resistant to its own antibiotic
and exemplarily was studied with regard to roseoflavin resistance, an issue,
which is crucial for developing flavin analogs as anti-infectives. S.
davawensis contains an
FMN riboswitch which is not affected by RoFMN and which was found to be
responsible for roseoflavin resistance ( color:windowtext;text-decoration:none">9). Moreover, a roseoflavin
exporter appears to be present which confers roseoflavin resistance as well.

Although, until now, only very few vitamin
analogs with antibiotic function have been identified, we expect that a
multitude of yet unknown vitamin analogs exists. These compounds could help to
replenish the arsenal of antimicrobials urgently needed to fight multiresistant
bacterial pathogens.

References

normal">1.         Pedrolli, D. B., Jankowitsch, F.,
Schwarz, J., Langer, S., Nakanishi, S., Frei, E., and Mack, M. (2013) Curr
Pharm Des 19, 2552-2560

normal">2.         Otani, S., Matsui, K.,
and Kasai, S. (1997) Osaka City Med J 43, 107-137

normal">3.         Jankowitsch, F., Kuhm, C., Kellner, R.,
Kalinowski, J., Pelzer, S., Macheroux, P., and Mack, M. (2011) J Biol Chem
286, 38275-38285

normal">4.         Hemberger, S., Pedrolli, D. B., Stolz, J.,
Vogl, C., Lehmann, M., and Mack, M. (2011) BMC Biotechnol 11,
119-129

normal">5.         Grill, S., Busenbender, S., Pfeiffer, M.,
Kohler, U., and Mack, M. (2008) J Bacteriol 190, 1546-1553

normal">6.         Langer, S., Nakanishi, S., Mathes, T.,
Knaus, T., Binter, A., Macheroux, P., Mase, T., Miyakawa, T., Tanokura, M., and
Mack, M. (2013) Biochemistry 52, 4288-4295

normal">7.         Mansjo, M., and
Johansson, J. (2011) RNA Biol 8, 674-680

normal">8.         Ott, E., Stolz, J.,
Lehmann, M., and Mack, M. (2009) RNA Biol 6, 276-280

normal">9.         Pedrolli, D. B., Matern,
A., Wang, J., Ester, M., Siedler, K., Breaker, R., and Mack, M. (2012) Nucleic
Acids Res 40, 8662-8673

normal">10.       Macheroux, P., Kappes,
B., and Ealick, S. E. (2011) Febs J 278, 2625-2634

normal">11.       Langer, S., Hashimoto,
M., Hobl, B., Mathes, T., and Mack, M. (2013) J Bacteriol 195,
4037-4045

normal">12.       Pedrolli, D. B.,
Nakanishi, S., Barile, M., Mansurova, M., Carmona, E. C., Lux, A., Gartner, W.,
and Mack, M. (2011) Biochem Pharmacol 82, 1853-1859

normal">13.       Serganov, A., Huang, L.,
and Patel, D. J. (2009) Nature 458, 233-237

Figure 1. The antibiotic/vitamin
analog roseoflavin from Streptomyces davawensis has multiple cellular
targets which may also be true for vitamin analogs yet to be discovered. Roseoflavin
is studied in our laboratory as a model compound. Roseoflavin enters the cell via
riboflavin transporters and is activated to RoFMN and RoFAD by flavokinases (EC
2.7.1.26) and FAD synthetases (EC 2.7.7.2), respectively. RoFMN blocks FMN
riboswitches, genetic elements, that regulate expression of genes responsible
for riboflavin biosynthesis and transport (13). In addition, RoFMN and RoFAD inhibit the activity of flavoenzymes
(FMN or FAD dependent), which are involved in a large variety of cellular processes
(10). As an example Escherichia coli is thought to contain 53
different flavoenzymes.