Crystal Structure, Modeling, and Identification of Key Residues Provide Insights into the Mechanism of the Key Toxoflavin Biosynthesis Protein ToxD

Toxoflavin,
a toxic secondary metabolite produced by a variety
of bacteria, has been implicated as a causative agent in food poisoning
and a virulence factor in phytopathogenic bacteria. This toxin is
produced by genes encoded in the tox operon in Burkholderia glumae, in which the encoded protein,
ToxD, was previously characterized as essential for toxoflavin production.
To better understand the function of ToxD in toxoflavin biosynthesis
and provide a basis for future work to develop inhibitors of ToxD,
we undertook the identification of structurally and catalytically
important amino acid residues through a combination of X-ray crystallography
and site directed mutagenesis. We solved the structure of BgToxD, which crystallized as a dimer, to 1.8 Å resolution.
We identified a citrate molecule in the putative active site. To investigate
the role of individual residues, we used Pseudomonas
protegens Pf-5, a BL1 plant protective bacterium known
to produce toxoflavin, and created mutants in the ToxD-homologue PFL1035.
Using a multiple sequence alignment and the BgToxD
structure, we identified and explored the functional importance of
12 conserved residues in the putative active site. Eight variants
of PFL1035 resulted in no observable production of toxoflavin. In
contrast, four ToxD variants resulted in reduced but detectable toxoflavin
production suggesting a nonessential role. The crystal structure and
structural models of the substrate and intermediate bound enzyme provide
a molecular interpretation for the mutagenesis data.