A surface‐exposed, network substitution results in a gain‐of‐function in tryptophan synthase6.5.0

Abstract

Tryptophan synthase (TS) is a α2β2 dimeric enzyme that catalyzes the final two reactions in the production of tryptophan. The product of the alpha subunit (αTS), indole, is channeled to the beta subunit (βTS) where it is then condensed with serine to form tryptophan. TS is a valuable model system for understanding enzyme allostery, and specifically enzyme‐enzyme communication. We previously identified amino acid interaction networks in αTS using NMR‐based methods. We proposed that these network residues play an important role in catalysis and communication between the two subunits. Based on previous studies, αTS residue Ala198 was identified as a residue of interest. Ala198 is a surface‐exposed, network residue that is dynamic on the ms‐μs timescale, according to our previous NMR studies. To better understand the networks, we characterized the A198W variant by performing cell‐based assays, kinetic assays, molecular dynamics (MD) simulations, and NMR experiments. We found that E.coli cells expressing the A198W variant had a faster growth rate in minimal medium compared to cells expressing the wild‐type αTS. Our kinetic studies on purified TS indicated that the A198W variant had little effect on αTS activity, but increased the activity of the TS complex (i.e. from initial indole‐3‐glycerol phosphate substrate to final tryptophan product), perhaps due to an enhancement of indole channeling. MD simulations indicated that the A198W substitution led to structural dynamic changes that propagated throughout αTS, including several key residues at the α/β binding interface. The MD simulations were consistent with the indole channel being more open, more often, which could help explain the increase in TS activity in vitro and in vivo. NMR experiments also indicated that the A198W substitution induced structural dynamic changes throughout αTS, consistent with the MD simulations. Despite Ala198 being distal from both the active site of αTS (27Å) and the α/β binding interface (30Å), substitutions at this position result in changes to catalytic efficiency and the TS complex dynamics. These findings underscore the importance of network residues on enzyme dynamics, allostery, and subunit communication. Engineering of surface‐exposed network residues represents one way of controlling enzyme activity.6.5.0Support or Funding Information6.5.06.5.0