Altering Active-Site Loop Dynamics Enhances Standalone\nActivity of the Tryptophan Synthase Alpha Subunit

The α-subunit (TrpA) of the allosterically regulated\nbifunctional\ntryptophan synthase αββα enzyme catalyzes\nthe retro-aldol cleavage of indole-glycerol phosphate (IGP) to d-glyceraldehyde 3-phosphate (G3P) and indole. The activity\nof the enzyme is highly dependent on the β-subunit (TrpB), which\nallosterically regulates and activates TrpA for enhanced function.\nThis contrasts with the homologous BX1 enzyme from Zea mays that can catalyze the same reaction as TrpA\nwithout requiring the presence of any additional binding partner.\nIn this study, we computationally evaluated and compared the conformational\nlandscapes of the homologous <i>Zm</i>BX1 and <i>Zm</i>TrpA enzymes. Our results indicate that enhanced TrpA standalone\nactivity requires the modulation of the conformational dynamics of\ntwo relevant active-site loops, loop 6 and 2, that need to be synchronized\nfor accessing the catalytically activated closed state for IGP cleavage,\nas well as open states for favoring indole/G3P release. Taking as\ninspiration the evolutionary blueprint <i>Zm</i>BX1 and\nusing our developed correlation-based tool shortest path map focused\non the rate-determining conformational transition leading to the catalytically\nactivated closed state, we computationally designed a variant named <i>Zm</i>TrpA<sup>SPM4‑L6BX1</sup>, which displays a 163-fold\nimprovement in catalytic efficiency for the retro-aldol cleavage of\nIGP. This study showcases the importance of fine-tuning the conformational\ndynamics of active-site loops for altering and improving function,\nespecially in those cases in which a conformational change is rate\ndetermining.