Violacein Biosynthesis Research Articles

Biosynthesis of violacein: intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side.

Feeding experiments with a mixture of [2-13C]- and [indole-3-13C]tryptophans, of [3-13C]- and [indole-3-13 C]tryptophans (1:1 molar ratio) and of others have proved that the 1,2-shift of the indole ring occurred via an intramolecular process for formation of the left part (5-hydroxyindole side) of the violacein skeleton and demonstrated that the C-C bond from C2 of the indole ring to C2 of the side chain was completely retained for formation of the right part (oxindole side) during the entire biosynthetic process. Due to the involvement of transaminase, it has remained unresolved whether indolylpyruvic acid is the biosynthetic intermediate and/or from where the nitrogen atom of the pyrrolidone ring originates. An incorporation experiment with a mixture of [2-13C]- and [alpha-15N]tryptophans (1:1 molar ratio) verified that the nitrogen atom in the central ring was exclusively derived from the right-side tryptophan. Thus, all the carbon and nitrogen atoms in the right part of the violacein skeleton were constructed by intact incorporation of the tryptophan molecule, with decarboxylation probably occurring at a later biosynthetic stage.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

ChemInform Abstract: Studies on the Biosynthesis of Violacein. Part 9. Green Pigments Possessing Tetraindole and Dipyrromethene Moieties, Chromoviridans and Deoxychromoviridans, Produced by a Cell‐Free Extract of Chromobacterium violaceum and Their Biosynthetic Origins.

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Studies on the biosynthesis of violacein. Part 9.1 Green pigments possessing tetraindole and dipyrromethene moieties, chromoviridans and deoxychromoviridans, produced by a cell-free extract of Chromobacterium violaceum and their biosynthetic origins

Novel green-coloured pigments, 5 and 6, are reported, which are concomitantly produced under a cell-free system together with the pigments of violaceins 1, 2 and proviolaceins 3, 4. The metabolites 5 and 6 have been named deoxychromoviridans and chromoviridans, respectively. The structures of 5 and 6 were established by analyses of the FABMS and various types of 2D-NMR spectra as 3-[2-{(Z)-[3,5-di(1H-indol-3-yl)-1H-pyrrol-2-yl]methylidene}-5-(1H-indol-3-yl)-2H-pyrrol-3-yl]-1H-indole and 3-[2-{(Z)-[3,5-di(1H-indol-3-yl)-1H-pyrrol-2-yl]methylidene}-3(1H-indol-3-yl)-2H-pyrrol-5-yl]-1H-indol-5-ol, respectively, and are closely related to those of violaceins 1, 2 and proviolaceins 3, 4, although they have an extra C1 unit at the dipyrromethene moiety and were not oxygenated at any position. The C1 unit of the dipyrromethene moiety was demonstrated to come from serine by feeding labelled [3-13C]serine, indicating that serine hydroxymethyltransferase and methylenetetrahydrofolate are responsible for the biosyntheses. The green pigments also have metal chelation capability with iron, copper, zinc and cobalt ions.

Biosynthesis of violacein: oxygenation at the 2-position of the indole ring and structures of proviolacein, prodeoxyviolacein and pseudoviolacein, the plausible biosynthetic intermediates of violacein and deoxyviolacein

During a search for biosynthetic intermediates of violacein pigments 1 and 2, employing enzyme inhibitors and blocked mutants, three novel tryptophan metabolites 3, 4 and 6 were found. Compounds 3 and 4, named prodeoxyviolacein and proviolacein, respectively, were red in neutral or basic medium, and blue in acidic solution. However, the red–orange colour of compound 6, named pseudoviolacein, did not change with acidity. The structures were determined by chemical modifications and by the analysis of HRMS (EI) and 2D NMR spectra resulting from COSY (1H–1H and 1H–13C), HMBC and NOESY experiments. The structures of 3, 4 and 6 were established as 3,5-di(indol-3-yl)pyrrol-2(2H)-one, 5-(5-hydroxyindol-3-yl)-3-(indol-3-yl)pyrrol-2(2H)-one and 5-(5-hydroxy-2-oxoindol-3-ylidene)-3-(indol-3-yl)pyrrol-2(1H)-one, respectively. The structure of 6 is similar to that of violacein 2, differing only in that the other indolyl group is oxygenated. Compounds 3 and 4 do not have an oxoindole unit. The isolation of 3, 4 and 6 together with 5(named as pseudodeoxyviolacein, previously published) lead to the proposition that the oxygenation at the 2-position of indole ring is a final step in the biosynthesis of violacein analogues.

Biosynthesis of a trypanocide by Chromobacterium violaceum

Radio-isotope studies indicated not only that L-tryptophan can serve as carbon source for synthesis of the trypanocide, violacein by Chromobacterium violaceum (BB-78 strain) but also that isatin and indole 3-acetic acid are both important metabolic intermediates. Using 3-indolyl [2-(14)C] and [1-(14)C] acetic acid, it was found that the carboxylic carbon was not eliminated and that indole-3-acetic acid was incorporated intact into the pigment structure. N-Ethyl(5-hydroxy-indol-3-yl)-2-indolylethylamide is also an important metabolic intermediate in the violacein biosynthesis. This is the first report of a metabolic scheme for violacein synthesis which includes an intermediate other than L-tryptophan.

A New Metabolite of Tryptophan, Chromopyrrolic Acid, Produced byChromobacterium violaceum

In order to trap the biosynthetic intermediate of violacein, a number of mutants were prepared. A novel tryptophan metabolite was produced by the administration of tryptophan to washed whole cells of the mutant of Chromobacterium violaceum. The chemical structure was determined to be 3,4.di.(3-indolyl)-pyrrole 2,5-dicarboxylic acid on the bases of 1H- and 13C-NMR spectra, and MS data. This compound was named chromopyrrolic acid, and was also detected in the various metabolites produced by the parent strain, although the amount produced was somewhat smaller. The structure of chromopyrrolic acid was closely related to that of violacein and deoxyviolacein. The pyrrole dicarboxylic acid moiety of chromopyrrolic acid was biosynthesized by condensation reactions of the tryptophan side chains of two tryptophan molecules, as was found in the biosynthesis of violacein. However, the 1,2-shift of the indole ring that was found in the biosynthesis of violacein did not occur. To confirm whether or not chromopyrrolic acid was a biosynthetic intermediate, a feeding experiment with radiolabeled chromopyrrolic acid was carried out. The incorporation into violacein analogues failed, indicating that chromopyrrolic acid was produced independently of violacein biosynthesis.

Studies on the biosynthesis of violacein. Part III. Biosynthesis of violacein: Evidence for the intermediacy of 5-hydroxy-L-tryptophan and the structure of a new pigment, oxyviolacein, produced by the metabolism of 5-hydroxytryptophan.

The administration of 5-hydroxy-L-tryptophan to growing cells yielded a new blue pigment. The chemical structure was determined to be 5-(5-hydroxyindol-3-yl)-3-(5-hydroxy-3-oxoindolylidene)-4-pyrroline-2-one, mainly by using proton- and carbon 13-NMR. Feeding experiments of 5-hydroxytryptophan labeled with deuterium and carbon-13 unambiguously demonstrated that the hydroxylation of tryptophan was the first step for violacein biosynthesis. No incorporation of 5-hydroxy-[4, 6-2H2]tryptamine suggests that decarboxylation of 5-hydroxy-L-tryptophan was not the second reaction, but should take place at a later stage in the pathway of violacein formation. 5-Hydroxy-[4, 6-2H2]indole was not incorporated, indicating that an enzyme like tryptophanase was unlikely to have been involved in violacein biosynthesis.

Studies on the biosynthesis of violacein. Part II. Biosynthesis of violacein: Origins of hydrogen, nitrogen and oxygen atoms in the 2-pyrrolidone nucleus.

The biosynthetic origins of the hydrogen, nitrogen and oxygen atoms in the pyrrolidone ring of violacein'were established by an anaylses of the 1H, 13C NMR and MS spectra of its isotope-enriched metabolites. Feeding experiments of [2-2H] and [3-2H2]tryptophans have revealed that the hydrogen in the pyrrolidone ring was derived from the methylene protons at the 3-position of tryptophan. The stereochemical fate of the prochiral hydrogens was determined to be in the retention of the pro-S hydrogen by these feeding experiments using [3R-2H] and [3S”-2H]tryptophans. The incorporation experiment of [α-15N]tryptophan demonstrated that the nitrogen atom in the ring originated from the α-amino group of tryptophan. The incorporation experiment of 18O2 gas verified that all the oxygen atoms of violacein were derived from the molecular oxygen.

Studies on the biosynthesis of violacein. part I Biosynthesis of violacein : A novel rearrangement in tryptophan metabolism with a 1,2-shift of the indole ring.

Studies on the biosynthesis of violacein. part I Biosynthesis of violacein : A novel rearrangement in tryptophan metabolism with a 1,2-shift of the indole ring.

Physiological aspects of violacein biosynthesis in nonproliferating cells.

Physiological aspects of violacein biosynthesis in nonproliferating cells.