While the study provides significant structural insights, there are some gaps in the exploration of functional dynamics. For example, the kinetic assays focus on steady-state parameters (e.g., k_cat/K_m) but do not address how conformational changes in the ATEYS loop dynamically regulate substrate access. Could the authors discuss whether time-resolved methods like stopped-flow spectroscopy or molecular dynamics simulations were considered to link structural flexibility with enzymatic kinetics? Additionally, the study demonstrates enhanced reactivity of the E493A mutant toward non-native substrates, yet it stops short of exploring its structural basis. Were cryo-EM or crystallographic data obtained for this mutant to confirm whether the altered activity corresponds to conformational changes?
The comment raises insightful questions about the connection between structural dynamics and enzymatic function in the study. In particular, the suggestion to explore time-resolved methods like stopped-flow spectroscopy or molecular dynamics simulations could significantly enhance the understanding of how conformational changes in the ATEYS loop regulate substrate access. Additionally, investigating the structural basis for the altered activity of the E493A mutant using cryo-EM or crystallography would complement the kinetic findings and provide a deeper mechanistic perspective.
From my understanding, the study’s focus on steady-state parameters and structural analysis of the wild-type enzyme provides a strong foundation, but it may have been limited by resource availability or technical constraints in performing advanced time-resolved or mutant-specific structural studies. Could the authors confirm if these aspects were considered but excluded due to practical limitations, or if they are being planned as part of future work?