Synthesis of (S)- and (R)-β-Tyrosine by Redesigned Phenylalanine Aminomutase

Round 1

Reviewer 1 Report

Dear Authors,
The article is clear and well-edited.
The theme is opportune and usefull.
There are only a few things to improve.

"Arg325" is missing from Figure 3. Please make up.

An interpretation of Figure 4 is missing from the article. It would be worth dedicating a paragraph to it.

Please insert the term "(Figure 6C)" in line 185.

Please insert the term "(Figure 6D)" in line 190.

Author Response

We hereby thank the reviewer for his/her comments.

The missing label Arg325 has been added in Figure 3.

The section from Line 116 to 129 is the explanation about Figure 4. We added “As shown in Figure 4B” in Line121 and “(Figure 4A)” in Line 125 to help readers to follow the text and figures. “Similarly, Leu104, Lys427, Ile431, and Glu455 from the binding site could be considered as the important residues for substrate selectivity.” has been added in Line 129 to point out more detail about the highly conserved residues in the active site.

In Line 189, “(Figure 6C) has been added.

In Line 190, “(Figure 6D) has been added.

Reviewer 2 Report

I have read the work on the synthesis of amino acids using modified phenylalanine aminomutase from Taxus chinensis. The work concerns an important aspect related to the selectivity and activity of this enzyme. The authors proposed a possible mechanism of action of this enzyme. They supported their hypothesis with numerous experiments involving the change of residues in the active center of the enzyme. The work is very well designed and the results presented convincingly. In my opinion, the work deserves publication in its current form. All experimental procedures are well described. The only remark concerns the poor quality of the attached figures on the protein structure

Author Response

We hereby thank the reviewer for his/her comments.

The attached figures on the protein structure (Figure 6, 7, 8) have been replaced by high-quality figures.

Reviewer 3 Report

The manuscript by Rudat and co-workers describes the use of modified phenylalanine aminomutase for the synthesis of (S)-ß and (R)-ß-tyrosine. Leu104 and its mutant Leu104Ser control the re-addition of ammonia on ß-position, thus forming (R)-ß-phenylalanine and (S)-ß-phenylalanine respectively. Previously authors reported the production of ß-tyrosine in lignin valorization through computationally designed enzyme (Catalysts 2021, 11(11), 1310). In the present study the demonstrated the necessity of conserved residue Leu179 for the activity, influence of the neighboring residues and stabilization of the carboxylate binding. Overall, the present work has sufficient novelty elements for publication and is presented nicely, therefore, this manuscript can be accepted for publication in Catalysts.

Comments:

I would suggest studying the temperature dependency of mutase activity.

In figure 3, label of Arg325 is missing.

Author Response

We hereby thank the reviewer for his/her comments.

To discuss about the relationship between mutase activity and temperature, the function of the residues on the inner loop in TchPAM has been added in table 2 with reference from Heberling, M.M, 2015. As well as sentences “Moreover, the reaction temperature could also be a key factor to influent the mutase activity of TchPAM by changing the inner loop structure (Pilbák, S 2006). It should be an attractive area in the future study on TchPAM.” have been added in Line 464 to 466 in Conclusion.

Pilbák, S.; Tomin, A.; Rétey, J.; Poppe, L. The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases. FEBS J. 2006, 273, 1004–1019, doi:10.1111/j.1742-4658.2006.05127.x.

Heberling, M.M.; Masman, M.F.; Bartsch, S.; Wybenga, G.G.; Dijkstra, B.W.; Marrink, S.J.; Janssen, D.B. Ironing out their differences: Dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase. ACS Chem. Biol. 2015, 10, 989–997, doi:10.1021/cb500794h

The label of Arg325 has been added in Figure 3.