Engineering of Microbial Substrate Promiscuous CYP105A5 for Improving the Flavonoid Hydroxylation
Round 1
Reviewer 1 Report
In this work, the authors achieved the identification, expression, biochemical characterization, and enzyme engineering of CYP105A5 from Streptomyces sp. They varied that CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A and regioselective C3'-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. They enhanced the product formation rate of flavonoids via rational design of substrate binding pocket based on the experimental data, homology modeling, and molecular docking analysis. The experiment results indicate that the double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. They also confirmed that the mutant’s kcat/Km values were increased by 1.68-fold and in vivo conversion rate was increased by 2.57-fold for naringenin. The result also demonstrated the future application of protein engineering to expand the substrate flexibility of enzymes towards flavonoids and/or other substrates.
In the introduction section, the authors stated that CYP105A5 was capable of oxyfunctionalization of diverse sets of flavonoids. However, I cannot find out detailed information about the catalytic performance of CYP105A5 in this section. Please give a brief review of the catalytic property of CYP105A5 for the hydroxylation of diverse flavonoids and the relevant references shall be listed in the revised version.
Author Response
Dear Editor-in-Chief and reviewers,
We highly appreciate the reviewer’s valuable time and response to our manuscript. We found the reviewer’s comments helpful in bringing the quality of our work. Therefore, we have made changes to our manuscript accordingly and to facilitate the review process of our revised manuscript, all revisions incorporated into the manuscript are marked up using the
tracking mode. Moreover, all revisions are also included in this reply and are highlighted in red color.
Reviewer #1
In this work, the authors achieved the identification, expression, biochemical characterization, and enzyme engineering of CYP105A5 from Streptomyces sp. They varied that CYP105A5 from Streptomyces sp. showed substrate flexibility with different flavonoids and was able to catalyze O-demethylation of biochanin A and regioselective C3'-hydroxylation of daidzein, genistein, and naringenin, and additional C8-hydroxylation for daidzein using heterologous redox partners putidaredoxin and putidaredoxin reductase. They enhanced the product formation rate of flavonoids via rational design of substrate binding pocket based on the experimental data, homology modeling, and molecular docking analysis. The experiment results indicate that the double mutant L100A/I302A and L100A/I408N exhibited greatly enhanced in vivo conversion rates for flavonoid hydroxylation. They also confirmed that the mutant’s kcat/Km values were increased by 1.68-fold and in vivo conversion rate was increased by 2.57-fold for naringenin. The result also demonstrated the future application of protein engineering to expand the substrate flexibility of enzymes towards flavonoids and/or other substrates.
In the introduction section, the authors stated that CYP105A5 was capable of oxyfunctionalization of diverse sets of flavonoids. However, I cannot find out detailed information about the catalytic performance of CYP105A5 in this section. Please give a brief review of the catalytic property of CYP105A5 for the hydroxylation of diverse flavonoids and the relevant references shall be listed in the revised version.
Response:
This is the first time that we are characterizing the CYP105A5. This enzyme was capable of hydroxylation and demethylation of flavonoids. There is no report of other enzymes from the CYP105A subfamily (like CYP105A1, CYP105A2, CYP105A3, CYP105A4, etc.) that can metabolize the flavonoid, till now. So, by stating that CYP105A5 was capable of oxyfunctionalization of diverse sets of flavonoids, we were trying to provide a summary of the present work in the last paragraph of the introduction section.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This manuscript by Oh et al. reports the cloning, expression, and engineering of a promiscuous CYP105A5 for the hydroxylation of flavonoid. Overall, it is well-written, and the results are interesting. I have a few comments for this manuscript as follows:
1. It is unclear why they used only site directed mutagenesis to construct the mutants, for example, for residue R85, only R85D was constructed and investigated, how about the other mutations?
2. For substrate Daidzein, both 3’-hydroxylated product and 8-hydroxylated product were produced, what is the ratio of these two products? And whether the mutants change the product ratio?
3. Line 329-330, please give some comments on this phenomenon
4. Line 23, please specify which mutant L100A/I302A or L100A/I408N
5. Line 116, “isoflavone” should read “isoflavones”
6. The ratio of P450/Pdx/Pdr was 1:8:2, please explain for this?
7. I100A/I408N is missing from Table 1
8. Line 267, “has been” should read “have been”
9. Line 299, “stearic hindrance” should read “steric hindrance”
10. Line 318, “excess channel” should read “access channel”
11. The gene ID for CYP105A5 should be provided
12. What exactly are the reaction time and temperature for enzyme activity assay? In line 403, it says 10 min at 37 oC, while in line 408, it says 1 h at 30 oC?
13. SI, Figure S4C, I think “P2” should be “S”
Author Response
Dear Editor-in-Chief and reviewers,
We highly appreciate the reviewer’s valuable time and response to our manuscript. We found the reviewer’s comments helpful in bringing the quality of our work. Therefore, we have made changes to our manuscript accordingly and to facilitate the review process of our revised manuscript, all revisions incorporated into the manuscript are marked up using the
tracking mode. Moreover, all revisions are also included in this reply and are highlighted in red color.
Reviewer #2
This manuscript by Oh et al. reports the cloning, expression, and engineering of a promiscuous CYP105A5 for the hydroxylation of flavonoid. Overall, it is well-written, and the results are interesting. I have a few comments for this manuscript as follows:
- It is unclear why they used only site directed mutagenesis to construct the mutants, for example, for residue R85, only R85D was constructed and investigated, how about the other mutations?
Response: We have mentioned that the amino acids were selected based on the analysis of docked substrate contact with the active site residues for the mutational study. The bulky hydrophobic amino acids (L100, L108, L252, I302) were converted to simple hydrophobic amino acid alanine. And for the two remaining amino acids, R85 and I408, based on the docking analysis of the wild-type and mutant protein, the particular mutants were chosen. The change of amino acid from basic to acidic (R85D) and from hydrophobic to basic (I408N) showed a significant change in the binding mode of the substrate and thus were selected for further study. We are also aware of other methods like alanine scanning and saturation mutagenesis. These methods require the generation of many mutants, and their screening is also very time-consuming. By creating a small set of mutants using site-directed mutagenesis we were successfully able to increase the substrate conversion rate.
- For substrate Daidzein, both 3’-hydroxylated product and 8-hydroxylated product were produced, what is the ratio of these two products? And whether the mutants change the product ratio?
Response: Agree and changes have been made.
The result showed that the 8-hydroxylated and 3'-hydroxylated products of daidzein represented 43% and 57% of the product formed. The mutants constructed were also unable to change the product distribution pattern. And these results are incorporated in the manuscript (line 129 and line 193-195) and supporting information (Figure S7).
Line 129: Likewise, the HPLC analysis of the daidzein showed two monohydroxylated products P1 and P2, representing 43% and 57% of the total product formed respectively (Figure S4B).
Line 193-195: Mutant L100A/I408N increased the daidzein hydroxylation activity but it was unable to change the product distribution pattern. Similar product distribution patterns were observed for all mutants (Figure S7).
Figure S7. The product distribution of wild-type and mutants of CYP105A5 catalyzed hydroxylation of daidzein. Biotransformations were performed using 3 μM purified P450 with CYP:Pdx:PdR at a ratio of 1:8:2 and analyzed by HPLC. The two hydroxylated products formed; 8-hydroxydaidzein and 3'-hydroxyaidzein are represented by yellow and green colors respectively.
- Line 329-330, please give some comments on this phenomenon
Response: For the CYP-dependent catalysis, electrons are provided by NAD(P)H and transferred to the heme cofactor via different redox partners. Electron delivery and proton flow are crucial for the efficient conversion of substrate to product. P450cam was efficiently able to utilize the electrons transferred through Pdx/PdR generated by oxidation of NADH. Instead of completing the catalysis cycle, if the highly active iron-oxygen complex decays at different stages, superoxide, hydrogen peroxide, or water are produced. This indicates the inability of CYP to efficiently utilize the generated electrons resulting in uncoupled reactions.
- Line 23, please specify which mutant L100A/I302A or L100A/I408N
Response: Agree and changes have been made.
Mutant L100A/I302A was missing in the sentence (line 23). Which has been corrected.
- Line 116, “isoflavone” should read “isoflavones”
Response: Agree and changes have been made. (Line 117)
- The ratio of P450/Pdx/Pdr was 1:8:2, please explain for this?
Response: The cytochrome P450 requires the redox partners putidaredoxin (Pdx) and putidaredoxin reductase (PdR) for the catalysis. PdR receives two electrons from NADH and delivers these to Pdx which in turn donates electrons to the cytochrome P450 enzyme for catalysis. The different ratios of these three proteins P450, Pdx, and Pdr are used during the reaction. For our study, we used the P450/Pdx/Pdr ratio of 1:8:2 which is clearly mentioned in the method section, line 407 (for enzyme activity assay: 3 µM CYP105A5, 24 µM Pdx, and 6 µM PdR) and line 430 (for determining kinetic parameters: 1 µM CYP105A5, 8 µM Pdx, and 2 µM PdR). We have also included the concentrations of enzymes used along with the ratio in the manuscript on line 202, line 224-225, and line 235-236, which were missing.
line 202: Activities are determined for purified CYP105A5 and its mutants using the CYP:PDX:PDR of 1:8:2 (3 µM CYP, 24 µM Pdx, and 6 µM PdR) and 0.2 mM substrate.
line 224-225: The overall apparent kinetic parameters were determined with CYP:Pdx:PdR concentration ratios of 1:8:2 (1 µM CYP, 8 µM Pdx, and 2 µM PdR) for purified CYP105A5 and its mutants towards daidzein, genistein, and naringenin.
line 235-236: The reaction mixture consisted of CYP:Pdx:Pdr in the ratios 1:8:2 (1 µM CYP, 8 µM Pdx, and 2 µM PdR) and in the presence of varied substrate concentrations.
- I100A/I408N is missing from Table 1
Response: Agree and changes have been made.
Due to the typographical error, the mutant I408N was typed as I408A. The correction has been done in table 1 and line 259.
- Line 267, “has been” should read “have been”
Response: Agree and changes have been made. (Line 270)
- Line 299, “stearic hindrance” should read “steric hindrance”
Response: Agree and changes have been made. (Line 302)
- Line 318, “excess channel” should read “access channel”
Response: Agree and changes have been made. (Line 321)
- The gene ID for CYP105A5 should be provided
Response: We have deposited our sequence in NCBI for gene ID but there is no response till now. We have provided the nucleotide sequence information of the CYP105A5 in the supporting information (Sequence S1). The nucleotide sequence of the CYP105A5 is acceptable, or it is mandatory to have an accession number. If it is mandatory, we have to wait till we obtain the accession number.
- What exactly are the reaction time and temperature for enzyme activity assay? In line 403, it says 10 min at 37 °C, while in line 408, it says 1 h at 30 oC?
Response: Agree and changes have been made.
All the enzyme activity assays were performed at 30 °C for 1 h. The sentence in line 406-407 was just a mistake and it has been removed.
- SI, Figure S4C, I think “P2” should be “S”
Response: Agree and changes have been made.
Author Response File: Author Response.pdf
