Expression, crystallization and preliminary crystallographic data analysis of VioD, a hydroxylase in the violacein-biosynthesis pathway

Abstract

Violacein, a natural purple secondary metabolite, is sequentially biosynthesized by five enzymes in the following pathway: VioA-VioB-VioE-VioD-VioC. VioD, a flavin-dependent oxygenase, catalyzes the hydroxylation of the intermediate product prodeoxyviolaceinic acid (PVA) at the 5-position of one indole ring to yield proviolacein. In vitro biochemical data have revealed this process, but the catalytic mechanism still remains largely unclear. Here, the cloning, expression, purification, crystallization and diffraction of VioD are reported. Crystals of VioD diffracted to 1.7 Å resolution and belonged to space group P31, with unit-cell parameters a = b = 90.0, c = 94.5 Å, α = β = 90, γ = 120°. Solvent-content calculation and molecular-replacement results suggest the presence of two molecules of VioD in the asymmetric unit.

Structural basis for substrate binding and catalytic mechanism of the key enzyme VioD in the violacein synthesis pathway

“…The full‐length vioD gene (GenBank KJ131413, amino acids 1–372) was cloned into the Nhe I/ Xho I sites of pET‐28a vector. PCR primers and template DNA used for DNA cloning are listed in Table 1, and the detailed cloning method is as before 31 . The residues sequences of the cloned genes were confirmed by DNA sequencing.…”

“…Commercial kits from Hampton Research and Microlytic (MCSG I–IV) were used for crystallization screening with protein concentration of 7.5 and 15 mg/mL. Single crystals were obtained from condition Index‐C1 (3.5 M sodium formate pH 7.0), 31 and MCSG I‐A6 (0.2 M ammonium sulfate, 0.1 M Bis‐Tris–HCl pH 5.5, 25% PEG 3350). Crystals grew to full size with dimensions of approximately 0.1 × 0.1 × 0.1 mm in 2 weeks.…”

“…Although several biochemical studies on the mechanisms underlying violacein biosynthesis have been reported, the detailed reaction mechanism of the VioD‐mediated conversion of PDVA to PVA remains to be elucidated. To elucidate the mechanism of VioD, we previously crystallized VioD and collected a 1.7 Å dataset 31 . Here, we solved VioD crystal structures to 1.7 Å (space group P6 4 ) and 2.1 Å (space group P2 1 2 1 2 1 ) resolution at two different space groups, respectively.…”

Advances in Chromobacterium violaceum and properties of violacein-Its main secondary metabolite: A review

“…It is an asymmetrical biomolecule that has a co-crystallized FAD molecule. VioD is a flavindependent oxygenase that is commonly responsible for the hydroxylation of prodeoxyviolaceinic acid (PVA) at the 5-position of the indole ring to yield proviolacein, and the structure this enzyme has been recently reported (Ran et al, 2015).…”

Multi-target drug with potential applications: violacein in the spotlight

“…VioDwhich is the enzyme capable of converting protodeoxyviolaceinic acid into protoviolaceinic acid from Duganella sp.-was expressed in E. coli. VioD was later purified and crystalized in order to generate X-ray diffraction (Ran et al 2015). VioA-which catalyzes the first step in violacein synthesis, i.e., l-tryptophan conversion into the corresponding α-imine-was also structurally and biochemically investigated to enable suggesting a mechanism for its activity (Füller et al 2016).…”