Enhancing the Thermal and Kinetic Stability of Ketol-Acid Reductoisomerase, a Central Catalyst of a Cell-Free Enzyme Cascade for the Manufacture of Platform Chemicals
, Yan Gao 3, Xiuna Yang 3, Ajit Kandale 1, Ross P. McGeary 1, Simon Williams 1, Bostjan Kobe 1,4, Mark A. Schembri 1,4, Michael J. Landsberg 1, Bin Wu 5, Thomas B. Brück 6, Volker Sieber 7, Mikael Boden 1, Zihe Rao 3, Sarel J. Fleishman 2, Gerhard Schenk 1,4,8 and Luke W. Guddat 1,*Round 1
Reviewer 1 Report
The paper “Enhancing the thermal and kinetic stability of ketol-acid reductoisomerase, a central catalyst of a cell-free enzyme cascade for the manufacture of platform chemicals” by Lv, Zhang, and colleagues deals with the engineering of a ketol-acid reductaseoisomerase to increase thermal stability using mutagenesis. This enzyme is of interest in the enzymatic synthesis of branched-chain amino acids. Oligomerization is also studied, as well as enzymatic performance in different conditions. A cryo-EM structure of a homododecamer of Class I KARI is also provided. The study is well conducted, the experimental design is sound and the authors’ conclusions are supported by the experimental data shown in the study. The work is suitable for publication in Applied Biosciences.
SPECIFIC COMMENTS
Figure 1 can be improved in quality. The delocalized electron density on the carboxylate is clipping in the central carbon. If possible, it would look better with a delocalized “arch”, instead of using three straight dashed lines.
Figure 5 – label descriptions as part of the figure itself would dramatically improve readability (color code for A and symbol code for B). The same applies to Figure 7.
Author Response
We are grateful for the reviewer's encouraging assessment of our study. He/she raised only a couple of minor points to be addressed:
1) Figure 1 can be improved in quality. The delocalized electron density on the carboxylate is clipping in the central carbon. If possible, it would look better with a delocalized “arch”, instead of using three straight dashed lines.
Response: we fixed this figure accordingly (see updated draft)
2) Figure 5 – label descriptions as part of the figure itself would dramatically improve readability (color code for A and symbol code for B). The same applies to Figure 7.
Response: Likewise, we also updated these two figures according to the reviewer's instructions (see updated draft).
Reviewer 2 Report
This is very interesting and exciting research. In this work, the author demonstrated that the oligomeric statues and interactions are critical to the stability of KARI enzyme. According to this, the author further computationally re-engineered EcKARI and built a variant with improved stability through by introducing new intermolecular interactions. This work should provide useful insights and methodology to design enzyme with enhanced stability.
I only have a smaller concern to this manuscript:
The author stated that: “This study demonstrated that the thermostability of KARI can be enhanced either by forming higher order oligomers or by in silico-guided mutagenesis.” However, no example on forming higher order oligomers to achieve improved thermostability was provided. Instead, the author demonstrated that by disassembling WT CjKARI from dodecameric to dimeric form, the stability of this enzyme is significantly reduced. Considering this, I would recommend a more solid conclusion: the oligomeric status is critical to the thermostability of KARI.
Author Response
We are also very happy about the strong endorsement from this reviewer. He/she had only one minor comment: "The author stated that: “This study demonstrated that the thermostability of KARI can be enhanced either by forming higher order oligomers or by in silico-guided mutagenesis.” However, no example on forming higher order oligomers to achieve improved thermostability was provided. Instead, the author demonstrated that by disassembling WT CjKARI from dodecameric to dimeric form, the stability of this enzyme is significantly reduced. Considering this, I would recommend a more solid conclusion: the oligomeric status is critical to the thermostability of KARI."
Response: In agreement with the reviewer's comment we changed the relevant sentence in the Conclusion from "This study demonstrated that the thermostability of KARI can be enhanced either by forming higher order oligomers or by in silico-guided mutagenesis" to "This study demonstrated that the oligomeric status is critical to the thermostability of KARI" (line 462 in revised draft).
