Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria

Gut microbial transformations of flavonoids, an enormous class of polyphenolic compounds abundant in plant-based diets, are closely associated with human health. However, the enzymes that initiate the gut microbial metabolism of flavones and flavonols, the two most abundant groups of flavonoids, as well as their underlying molecular mechanisms of action remain unclear. Here, we discovered a flavone reductase (FLR) from the gut bacterium, Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740), which specifically catalyses the hydrogenation of the C2–C3 double bond of flavones/flavonols and initiates their metabolism as a key step. Crystal structure analysis revealed the molecular basis for the distinct catalytic property of FLR. Notably, FLR and its widespread homologues represent a class of ene-reductases that has not been previously identified. Genetic and biochemical analyses further indicated the importance of FLR in gut microbial consumption of dietary and medicinal flavonoids, providing broader insight into gut microbial xenobiotic transformations and possible guidance for personalized nutrition and medicine.

Page 4, line 79: "in the gut F. plautii ATCC 49531", remove "the gut" or rephrase to "the gut bacteria, F. pla.." Other instances of this in the manuscript should also be addressed.
Page 18, line 358, "indicating that FLR and WrbA are far in evolution".Rephrase as it does not have the intended meaning.Also remember that structure rather than sequence is more indicative of evolutionary relatedness.Pairwise alignment values over the entire protein would probably be less confusing that percentage identity over a small coverage region and lead to less confusion.

Dear Reviewers,
We appreciate your valuable comments on the manuscript (NCOMMS-20-31553A).All the comments and suggestions have been very carefully considered, and the revised points are as listed below.

Reviewer #4 (Remarks to the Author):
In the manuscript entitled "Discovery of a novel class of ene-reductases for initiating flavonoid catabolism in gut bacteria", Yang et al describes the discovery of an enzyme capable of catalyzing the first reductions step of flavones and flavonols.Similar to other ene-reductases, it catalyzes the reduction of the conjugated C-C double bond on the heterocyclic C ring.The authors proved this through knockout and complementation studies in vivo, and also in vitro activity.The structure of the flavone reductase (FLR) were solved and kinetic analysis were also performed.
After reading the manuscript and the SI, I had some questions that was answered only by reading the rebuttal letter and comments to the reviewers from the previous round of reviewing.

I largely agree with these reviewers, and the authors should have included the additional data in their manuscript or SI, and not only in the point-to-point response (i.e. the growth curves showing the effects of the knockout, the bacterial IFR data, etc). I would strongly urge the authors to re-examine the additional data in the response to reviewers and include this in the SI and briefly referring to it in the main text, as I suspect most readers will have the same questions.
Reply: Thanks for the suggestion.We have re-checked the content in the response to reviewers and included the additional data in the Supplementary information section, including the growth curve of the flr-deleted and wild-type C. ljungdahlii strain (Supplementary Fig. 13), the effect of apigenin on the growth of each individual isolate in the microbial community constructed in this study (Supplementary Fig. 14), the activity assay of IFRs (sgrIFR, gene ID: SGR_2256) from Streptomyces griseus (Supplementary Fig. 1d and 1e), an extra sequence similarity network (SSN) with an increased edge score (Supplementary Fig. 7), and the possible mechanism of apigenin reduction by the FLR enzyme (Supplementary Fig. 15).

The title might be misleading, as this FLR are only responsible in flavone and flavonol metabolism and not all flavonoids subclasses as given in the introduction.
Reply: Thanks for the suggestion.The title has been modified to "Discovery of an ene-reductase for initiating flavone and flavonol catabolism in gut bacteria".

The low catalytic rate of this enzyme is disconcerting, but also supported in the in vitro
experiments, where it took the bacteria more than 24 h to convert 0.1 mM.
Reply: Thanks for the comments.Actually, this is also an issue of concern in the first round of reviewing.To confirm this result, we tested more FLR-like enzymes and added the data into the last round of revision (Supplementary Figure 6).A possible reason for the low catalytic rate of this enzyme is that the in vitro experiment is different from the real intestinal environment and therefore can not show the full potential of this enzyme.

Many parts of the figures in the main text can be moved to the SI, as some of it simply does not really add crucial insight.
Reply: According to the suggestion, Fig. 1b, Fig. 2a, and Fig. 2d have been moved to the SI (Supplementary Fig. 1a, Supplementary Fig. 3, and Supplementary Fig. 4a).

Also, some parts should be revised, especially the close up structures of the active site with the bound substrates in the main as well as the SI. The authors should include the OMIT maps of the co-factors and the substrates.
Reply: Thanks for the comments and suggestions.We have made corresponding changes to Fig.

In the proposed mechanism, the authors indicate a specific enantiomer forming. Was the chirality proven?
Reply: According to this suggestion, we have examined the chirality of the naringenin produced from FLR-catalyzed apigenin hydrogenation using circular dichroism (CD) spectrum.As expected, we obtained the CD spectrum of the naringenin product (as shown below, right figure), which is consistent with the previously reported CD spectra of S-(-)-naringenin (left figure).This result has been added into the revised manuscript (Supplementary Figure 16).

Please include CC1/2 values of data collection (SI Table)
Reply: The CC1/2 values of data have been added into the Supplementary Table 3.

I do not agree with the term "unorthodox" used in the revision
Reply: Thanks for pointing out this deficiency.The term "unorthodox" has been deleted or changed to "distinct" in the revised manuscript (line 28; line 104; line 167; line 978).The reported absorption (UV, thin line) and circular dichroism (CD, thick line) spectra of S-(-)-naringenin in ethanol.Org.Biomol. Chem., 2004, 2, 3602-3607 4c and Supplementary Fig.10, and added omit maps of the co-factors and substrates (as shown below).The main text has been modified accordingly (Line 242-243).

Fig. 4c ,
Fig. 4c, Zoom-in views of the cofactor FMN-and apigenin-binding site.The pictures show the amino acid residues surrounding the active site, and Fo-Fc density map(blue), contoured at 1.0 σ level, from which the FMN and apigenin were omitted.

Page 4 ,
line 79: "in the gut F. plautii ATCC 49531", remove "the gut" or rephrase to "the gut bacteria, F. pla.." Other instances of this in the manuscript should also be addressed.Reply: Thanks for the suggestion.This place (Page 4, line 79) and other similar instances in the manuscript have been revised.Page 18, line 358, "indicating that FLR and WrbA are far in evolution".Rephrase as it does not have the intended meaning.Also remember that structure rather than sequence is moreThe UV (black line) and circular dichroism (CD, red line) spectra of the separated S-(-)-naringenin in methanol solution.