The Violacein Biosynthetic Enzyme VioE Shares a Fold with Lipoprotein Transporter Proteins

Abstract

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. Our 1.21 Å resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB. For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE have identified the active site and five residues important for binding or catalysis. This structural and mutagenesis study suggests that VioE acts as a catalytic chaperone, using a fold previously associated with lipoprotein transporters to catalyze the production of its prodeoxyviolacein product.Violacein 5 (Fig. 1) is a purple pigment that gives tropical bacterium Chromobacterium violaceum its characteristic coloring (1-5). Violacein 5 has potential medical applications as an antibacterial (6 -8), anti-tryptanocidal (8 -10), anti-ulcerogenic (11), and anti-cancer drug (8,(12)(13)(14)(15)(16)(17). The molecule, made of L-tryptophan and molecular oxygen (18 -21), was originally thought to be synthesized by the action of just four enzymes VioABCD (22, 23), but an additional open reading frame (24), also in the operon, was shown to encode the enzyme VioE (25, 26). A 22-kDa protein with few homologues based on sequence analysis, VioE is required for formation of prodeoxyviolacein 3, a precursor to violacein 5 (25-27). Without the presence of VioE, a shunt product, chromopyrrolic acid 4, is produced, rather than 3 (25-27). VioE is therefore required for the 1,2 shift of the indole ring and subsequent chemistry that gives violacein 5 its distinctive architecture. Surprisingly, despite carrying out this chemistry, VioE lacks any bound cofactors or metal ions (26).Using genetic and biochemical analysis, an enzymatic pathway for violacein 5 production has been determined ( Fig. 1), and intermediates have been identified (26), with the major exception that the substrate of VioE (product of VioB) is still unknown. It has, however, been established that the product of VioB is the substrate for VioE; when VioA and VioB are incubated in a reaction mixture with the VioA substrate L-tryptophan for 10 min, passed through a 10-kDa filter (removing VioA and VioB), and then incubated with VioE, prodeoxyviolacein 3 is produced (26). By contrast, in the absence of VioE, no prodeoxyviolacein 3 is made (26). This result reveals three things: first, the VioB product is a small molecule that can pass through a 10-kDa filter; second, no interaction between VioB and VioE is necessary for product formation; and third, VioE is a catalyst. The small molecule passed through the filter (the product of VioB and substra...

A short helix in the C‐terminal region of LolA is important for the specific membrane localization of lipoproteins

“…RseB constitutes a signal transduction pathway leading to the induction of r E expression and is speculated to bind mislocalized lipoproteins to the hydrophobic cavity [20]. VioE is speculated to play a key role in the biosynthesis of violacein [21,22], a purple pigment with antibacterial and cytotoxic properties. Non-specific membrane interaction and discharge of cargos are unfavorable for the functions of these proteins although their exact functions remain to be clarified.…”

Pathway redesign for deoxyviolacein biosynthesis in Citrobacter freundii and characterization of this pigment

“…Despite this great interest, there has been a paucity of studies regarding the isolation of new bisindole molecules from natural sources or the synthesis of derivatives for comparison of their bioactivities and the study of bisindoles' mechanism(s) of action (Ryan et al 2008).…”

Biogenesis of Outer Membranes in Gram-Negative Bacteria

“…Not shown here, VioE (3BMZ, 2ZF3, 2ZF4) of Chromobacterium violaceum has a similar structure. VioE might play a key role in the biosynthesis of violacein, 69,70) a purple pigment with antibacterial and cytotoxic properties.…”