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A Common Late-Stage Intermediate in Catalysis by 2-Hydroxyethyl-phosphonate Dioxygenase and Methylphosphonate Synthase
- Peck, Spencer
C., Chekan, Jonathan R., Ulrich, Emily
C., Nair, Satish K., van der Donk, Wilfred A.
- Journal of the American Chemical Society 2015 v.137 no.9 pp. 3217-3220
- X-radiation, active sites, catalytic activity, chemical bonding, histidine, iron, isotopes, mutants, oxygenases
- 2-Hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS) are nonheme iron oxygenases that both catalyze the carbon–carbon bond cleavage of 2-hydroxyethylphosphonate but generate different products. Substrate labeling experiments led to a mechanistic hypothesis in which the fate of a common intermediate determined product identity. We report here the generation of a bifunctional mutant of HEPD (E176H) that exhibits the activity of both HEPD and MPnS. The product distribution of the mutant is sensitive to a substrate isotope effect, consistent with an isotope-sensitive branching mechanism involving a common intermediate. The X-ray structure of the mutant was determined and suggested that the introduced histidine does not coordinate the active site metal, unlike the iron-binding glutamate it replaced.