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.

毒黄素（Toxoflavin）是一类由多种细菌产生的有毒次级代谢产物，被认定为食物中毒的致病因子，同时也是植物病原细菌的毒力因子。该毒素由谷氏伯克霍尔德菌（Burkholderia glumae）的tox操纵子编码的基因合成，其编码的蛋白ToxD此前被证实为毒黄素合成所必需。为深入解析ToxD在毒黄素生物合成中的功能，并为后续开发ToxD抑制剂奠定研究基础，我们结合X射线晶体学（X-ray crystallography）与定点诱变（site directed mutagenesis）技术，鉴定了对结构和催化功能至关重要的氨基酸残基。我们解析了BgToxD的晶体结构，该蛋白以二聚体形式结晶，分辨率达1.8埃（Å）。我们在其推定的活性位点中发现了一个柠檬酸分子。为探究单个残基的功能角色，我们选用产毒黄素的植物保护性细菌产碱假单胞菌Pf-5（Pseudomonas protegens Pf-5），对其ToxD同源蛋白PFL1035构建了突变体。基于多序列比对与BgToxD的晶体结构，我们鉴定并探究了推定活性位点内12个保守残基的功能重要性。其中8个PFL1035突变体无法检测到毒黄素的合成；与之形成对比的是，4个ToxD突变体的毒黄素产量虽有所降低，但仍可被检测到，提示这些残基并非功能必需。底物与中间体结合状态下的晶体结构及结构模型，为诱变实验得到的数据提供了分子层面的合理解释。