Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA from Chromobacterium violaceum

Abstract

Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of L-tryptophan into the corresponding ␣-imine catalyzed by the flavoenzyme L-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA⅐FADH 2 and of the ternary VioA⅐FADH 2 ⅐2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of The amino acid tryptophan (Trp) is a versatile metabolite that is shunted into several microbial secondary pathways. The oxidative dimerization of two Trp scaffolds is an essential step for the synthesis of the natural bisindole compounds violacein, rebeccamycin, and staurosporine. The water-insoluble purple pigment violacein is mainly produced by the bacteria Chromobacterium violaceum and Janthinobacterium lividum in tropical habitats where it might be responsible for the shielding against UV radiation (1-3). Various biological activities of violacein, including antibacterial, antiviral, as well as cytotoxic effects against several tumor cell lines, have been demonstrated (4). The related compounds rebeccamycin and staurosporine show strong antitumorigenic activity because of DNA topoisomerase or protein kinase inhibition (5, 6).Synthesis of violacein, rebeccamycin, or staurosporine is based on a closely related initial pathway in which orthologous enzymes catalyze the oxidation of a Trp moiety into the related indole-3-pyruvic acid (IPA) 2 imine by the enzymes VioA, RebO, or StaO ( Fig. 1) (7-9). Subsequently, oxidative coupling of two imines by VioB, RebD, or StaD results in the formation of a short-lived compound that was proposed to be an IPA imine dimer (7, 10). For the synthesis of rebeccamycin and staurosporine, this reactive intermediate is spontaneously converted into chromopyrrolic acid (11-13). By contrast, violacein biosynthesis requires a key intramolecular rearrangement. The postulated IPA imine dimer is the substrate of VioE, which is catalyzing a [1,2]-shift of the indole ring to produce protodeoxyviolaceinic acid (7,14). Fig. 1 gives a schematic overview about the related pathways as follows: common enzymatic reactions and the involved cofactors are highlighted (gray shading), subsequent steps for the synthesis of violacein (VioE, VioD, VioC, and one autocatalytic step) (3, 7), rebeccamycin (RebP, RebC, RebG, and RebM) (15, 16), and staurosporine (StaP, StaC, StaG, StaN, StaMA, and StaMB) (16) are indicated.Synthesis of violacein starts with the FAD-dependent oxidation of L-Trp to IPA imine catalyzed by VioA. The VioA protein from C. violaceum shares a su...

A GenoChemetic strategy for derivatization of the violacein natural product scaffold

“…Multiple sequence alignment of 14 VioA homologues from various bisindole pathways that were characterized previously, shows that while Arg64 and Tyr309 are conserved among the VioA homologues, His163 is replaced by Asn in HysO and AbeO ( Figure S7), supporting the hypothesis that His163 is not required for catalytic activity of VioA in a previous study 14 . It would be interesting to compare the reaction kinetics of VioA homologues from other bisindole pathways, such as StaO (staurosporine pathway) 23 , EspO (erdasporine pathway) 37 and HysO …”

“…At the substrate-FAD interface, two key residues, Arg64 and Tyr309, were close to the charged carboxylate group of the substrate, positioning the Cα atom next to the FAD isoalloxazine plane ( Figure 4B). However, a previous study found that while Arg64 and Tyr309 are essential for catalytic activity, His163 is unnecessary as a VioA H163A mutant retained 85% of WT activity, suggesting that the hydride transfer from Cα and amine groups of Ltryptophan may not require acid-base catalysis of the H163 side chain 14 .…”

“…Surprisingly, C5-and C6-substituted tryptophan analogues tested in this study, such as 5-fluoro-tryptophan and 6-fluoro-tryptophan, were shown to have higher total analogues percentage composition than 4-fluoro-tryptophan ( Figure 2B). In addition, 5-methyl-tryptophan was shown to have higher affinity than Ltryptophan in a previous study 14 , suggesting that C5-substituted tryptophan analogues may bind to the VioA active site with alternative conformation which confers higher catalytic turnover, although further studies would be needed to validate this hypothesis.…”

“…A structure of VioA in complex with a competitive inhibitor 2-[(1H-indol-3-yl)methyl]prop-2-enoic acid (IEA) has been published independently (PDB accession code 5G3U) 14 , with slightly different crystallization conditions and crystallographic space group. In this study, all VioA active site mutants exhibited decreased activity with most tryptophan analogues tested compared to wild-type VioA, demonstrating the difficulty in rationally engineering VioA towards increased substrate promiscuity 14 .…”

“…compared to the cognate substrate L-tryptophan 14 , demonstrating potential for analogue generation using the violacein biosynthesis pathway.…”