Identification of the Key Enzyme of Roseoflavin Biosynthesis.

The bacteria Streptomyces davawensis and Streptomyces cinnabarinus produce roseoflavin, the only known natural riboflavin (vitamin B2 ) analogue with antibiotic activity. Roseoflavin can be considered a natural antimetabolite and has been postulated to be biosynthesized from riboflavin via the key intermediate 8-demethyl-8-aminoriboflavin (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. The pathway from riboflavin to AF has remained elusive, and the corresponding enzyme/s was/were unknown. Herein, we show by systematic gene deletion, heterologous gene expression, and biochemical studies that the enzyme specified by the gene BN159_7989 from S. davawensis is able to carry out a whole set of chemical reactions starting from riboflavin-5'-phosphate to give the final product 8-demethyl-8-aminoriboflavin-5'-phosphate (AFP).

Systemic intracellular analysis for balancing complex biosynthesis in a transcriptionally deregulated Escherichia coli l-Methionine producer.

l-Methionine (l-Met) has gained remarkable interest due to its multifaceted and versatile applications in the fields of nutrition, pharmaceuticals and clinical practice. In this study, the fluxes of the challenging l-Met biosynthesis in the producer strain Escherichia coli (E. coli) DM2853 were fine-tuned to enable improved l-Met production. The potential bottlenecks identified in sulfur assimilation and l-Met synthesis downstream of O-succinyl-l-homoserine (OSHS) were addressed by overexpressing glutaredoxin 1 (grxA), thiosulfate sulfurtransferase (pspE) and O-succinylhomoserine lyase (metB). Although deemed as a straightforward target for improving glucose-to-Met conversion, the yields remained at approximately 12%-13% (g/g). Instead, intracellular l-Met pools increased by up to four-fold with accelerated kinetics. Overexpression of the Met exporter ygaZH may serve as a proper valve for releasing the rising internal Met pressure. Interestingly, the export kinetics revealed maximum saturated export rates already at low growth rates. This scenario is particularly advantageous for large-scale fermentation when product formation is ideally uncoupled from biomass formation to achieve maximum performance within the technical limits of large-scale bioreactors.

Genome sequence of the bacterium Streptomyces davawensis JCM 4913 and heterologous production of the unique antibiotic roseoflavin.

Streptomyces davawensis JCM 4913 synthesizes the antibiotic roseoflavin, a structural riboflavin (vitamin B(2)) analog. Here, we report the 9,466,619-bp linear chromosome of S. davawensis JCM 4913 and a 89,331-bp linear plasmid. The sequence has an average G+C content of 70.58% and contains six rRNA operons (16S-23S-5S) and 69 tRNA genes. The 8,616 predicted protein-coding sequences include 32 clusters coding for secondary metabolites, several of which are unique to S. davawensis. The chromosome contains long terminal inverted repeats of 33,255 bp each and atypical telomeres. Sequence analysis with regard to riboflavin biosynthesis revealed three different patterns of gene organization in Streptomyces species. Heterologous expression of a set of genes present on a subgenomic fragment of S. davawensis resulted in the production of roseoflavin by the host Streptomyces coelicolor M1152. Phylogenetic analysis revealed that S. davawensis is a close relative of Streptomyces cinnabarinus, and much to our surprise, we found that the latter bacterium is a roseoflavin producer as well.

A novel N,N-8-amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces davawensis.

Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 µm; apparent K(D) for AF = 10.0 µm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.

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

UNLABELLED: 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/ß 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. DATABASE: Structural data are available in the Protein Data Bank under accession number 4D7K.

Natural riboflavin analogs.

Riboflavin analogs have a good potential to serve as basic structures for the development of novel anti-infectives. Riboflavin analogs have multiple cellular targets, since riboflavin (as a precursor to flavin cofactors) is active at more than one site in the cell. As a result, the frequency of developing resistance to antimicrobials based on riboflavin analogs is expected to be significantly lower. The only known natural riboflavin analog with antibiotic function is roseoflavin from the bacterium Streptomyces davawensis. This antibiotic negatively affects flavoenzymes and FMN riboswitches. Another roseoflavin producer, Streptomyces cinnabarinus, was recently identified. 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, thus allowing a further extension of the catalytic spectrum of flavoenzymes.

Riboflavin analogs as antiinfectives: occurrence, mode of action, metabolism and resistance.

Antimetabolites are molecules, 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 have a large variety of applications in the pharmaceutical and food industries. The identification of the structural riboflavin(vitamin B2) analog roseoflavin in Streptomyces davawensis demonstrates that anti-vitamins/cofactor analogs may serve as lead structures for the development of novel antibiotics. The latter is supported by the recent finding that roseoflavin had a profound inhibiting effect on the growth and infectivity of the human bacterial pathogen Listeria monocytogenes at very low concentrations. Roseoflavin is studied in our laboratory as a model compound. We investigate the biosynthesis, the possible large-scale production, the metabolization,the mechanism of action and the resistance mechanism of the producer organism in order to pave the way for the structured analysis of other vitamin analogs yet to be discovered. These compounds hopefully will help to replenish the arsenal of antimicrobials urgently needed to fight multiresistant bacterial pathogens.