Tryptophan synthase (TS) is a bienzyme complex responsible for catalyzing the last two reactions in the biosynthesis of tryptophan. The a subunit of TS (aTS) catalyzes the conversion of indole-3-glycerol phosphate (IGP) to glyceraldehyde-3-phosphate (G3P) and indole.  When aTS binds IGP, structural rearrangements occur centering around the �2a2 and �6a6 loops. Previous NMR assignments in both loops have been difficult due to exchange broadening. Here, we have completed the assignments of the 15N-labeled Ala resonances using titrations with G3P and indole, and site-directed mutagenesis. Mutations in the �2a2 loop lead to long-range structural changes in the �6a6 loop and the phosphate binding region, as assessed by 1H-15N HSQC chemical shift changes. Likewise, titration with G3P and/or indole lead to chemical shift changes in regions distant from the expected ligand binding sites. While the ps-ns timescale dynamics, assessed by 1H-15N heteronuclear NOE, of free aTS did not appear to be qualitatively different from aTS bound with G3P and/or indole, the �s-ms timescale dynamics, assessed by R2 relaxation dispersion, changed substantially depending on the ligand. For example, new �s-ms timescale dynamic motions were induced in the region around the indole (G3P) binding pocket when G3P (indole) were bound. Additional changes were observed in the presence of both ligands. In particular, Ala59, next to catalytic Glu60, displayed a conformational change when G3P and indole were both present, compared to indole alone; such structural rearrangements are likely necessary for chemical catalysis. Altogether, our results suggest that aTS is a dynamically rich enzyme where the �s-ms timescale dynamics are highly coordinated and are important for ligand binding, chemical catalysis, and likely, interaction with the � subunit of TS.

Structural and dynamic changes in the alpha subunit of tryptophan synthase important for catalysis and substrate channeling

246th National Meeting (2013)

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