Altering Active-Site Loop Dynamics Enhances Standalone Activity of the Tryptophan Synthase Alpha Subunit
Cristina Duran, Thomas Kinateder, Caroline Hiefinger, Reinhard Sterner, Sílvia Osuna
Abstract
The α-subunit (TrpA) of the allosterically regulated bifunctional tryptophan synthase αββα enzyme catalyzes the retro-aldol cleavage of indole-glycerol phosphate (IGP) to d -glyceraldehyde 3-phosphate (G3P) and indole. The activity of the enzyme is highly dependent on the β-subunit (TrpB), which allosterically regulates and activates TrpA for enhanced function. This contrasts with the homologous BX1 enzyme from Zea mays that can catalyze the same reaction as TrpA without requiring the presence of any additional binding partner. In this study, we computationally evaluated and compared the conformational landscapes of the homologous Zm BX1 and Zm TrpA enzymes. Our results indicate that enhanced TrpA standalone activity requires the modulation of the conformational dynamics of two relevant active-site loops, loop 6 and 2, that need to be synchronized for accessing the catalytically activated closed state for IGP cleavage, as well as open states for favoring indole/G3P release. Taking as inspiration the evolutionary blueprint Zm BX1 and using our developed correlation-based tool shortest path map focused on the rate-determining conformational transition leading to the catalytically activated closed state, we computationally designed a variant named Zm TrpA SPM4-L6BX1, which displays a 163-fold improvement in catalytic efficiency for the retro-aldol cleavage of IGP. This study showcases the importance of fine-tuning the conformational dynamics of active-site loops for altering and improving function, especially in those cases in which a conformational change is rate determining.