Can flavoprotein monooxygenases functionalize long-chain n-alkanes?

Raul Mireles; Arne Matthews; Robin Teufel; Lianet Noda-García

doi:10.1371/journal.pone.0332702

Peer Review History

Original SubmissionMay 21, 2025
13 Jun 2025 Decision Letter - Kshatresh Dutta Dubey, Editor -->PONE-D-25-27398-->-->Can flavoprotein monooxygenases functionalize long-chain n-alkanes?-->-->PLOS ONE Dear Dr. Noda-Garcia, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jul 28 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. --> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** -->2. Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: N/A Reviewer #2: Yes Reviewer #3: N/A ****** -->3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** -->4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** -->5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: This study reports on something that is often encountered, but is rarely published – a negative result. In this case, a fairly exhaustive attempt was made to replicate the prior, published observation that LadA has n-alkane monooxygenase activity in vitro. An array of assay conditions were attempted and multiple LadA homologs from other organisms were assessed. In all cases, no n-alkane monooxygenase activity was detected, suggesting that LadA-like enzymes are unable to catalyze the previously published reaction and the true function of LadA is still unknown. The fact that this was independently evaluated by two separate groups in this study (Noda-Garcia and Teufel groups) provides an additional level of confidence in the findings. This study appears to be well-conducted and provides information that will be useful for anyone studying LadA. However, I have one major suggesting for the authors before publishing this study: 1. Assess LadA for activity under conditions with MgSO4, with and without Fre, similar to the published studies, even though they do not seem mechanistically sound. I realize that there is no rationale for needing Mg2+ and a flavin reductase is usually necessary for these two component systems to function. In my experience, NADH can nonenzymatically reduce FMN, albeit poorly, so it is conceivable that FMNH2 could still be generated in the assay without Fre. I appreciate that the results of this additional experiment will very likely be the same as all of your others (no activity). Without it, though, a skeptical reader of your study may wonder if you didn’t observe activity with LadA simply because you opted not to use similar conditions to the the prior studies. Minor suggestion: 2. Be a little more forceful in pointing out how nonsensical some of the conditions are from the prior published studies. “In the study by Feng et al. (2007), the reaction setup included 1 mM hexadecane and 1 mM MgSO₄ in a 50 mM Tris-HCl buffer at pH 7.5, along with 1 mM NADH.” It is unclear how LadA activity could be detected in those experiments without FMN added since Group C FMOs usually do not purify with FMN bound. “The second study by Li et al. (2008), which focused on the structural analysis of LadA, also used 1 mM hexadecane in a 50 mM Tris-HCl buffer at pH 7.5; however, NADH was omitted, and 1 mM FMNH2 was added instead.” No information was provided to describe how the FMNH2 was generated for these experiments. This is critical information, as FMNH2 is not stable under aerobic conditions. Reviewer #2: This manuscript addresses the challenging issue of n-alkane functionalization. The authors have undertaken an effort to clarify the enzyme activity of the flavin-dependent enzyme LadA, an enzyme previously suggested to function as a long-chain n-alkane monooxygenase. Their rigorous experimentation, including testing optimized variants and novel homologs under in vitro conditions, significantly advances the current understanding. Our research group has also been particularly intrigued by LadA and pursued extensive studies aimed at reproducing the previously reported literature findings, unfortunately without success. Therefore, we highly appreciate the authors' systematic approach in clearly demonstrating and openly discussing the reproducibility challenges and limitations related to LadA activity. Their results provide crucial clarity and offer a balanced and critical evaluation of the previously reported roles of LadA and related flavoprotein monooxygenases. I congratulate the authors on this valuable and rigorous study, which strongly contributes to redefining the biochemical understanding of these enzymes. Their findings represent an essential milestone and open new avenues for research to uncover the true biochemical roles of LadA and its homologs. Reviewer #3: Dear Authors that is a very important finding and clarifies many open questions we had for this special FPMO, it also demonstrates how experimental setup can be designed to answer research questions from different perspectives. I have some minor mostly text-related suggestions which I hope help to improve the manuscript. Comments: - “sp.“ should read italics (e.g., page 3 “AlmA in Acinetobacter …” - “E. coli” should read italics - “(18 μM) LadA to our assays,,” comma issue to be corrected - Be consistent with “n-alkane” in terms of “n” reads italics - Protein expression is wrong; only genes can be expressed; correct throughout. - In your assays you provide Fre to provide reduced flavin for the potential monooxygenase; did you observe any H2O2 formation and if; was it different plus/minus substrate being present, that would at least indicate a ligand binding and thus effector role. - Fre might not be the best reductase partner as some two-component FPMOs need a certain partner to become active; like cooperativity and this might be an important conclusion to search for the natural redox couple. - If the potential uncoupling was high and the redFMN transfer not efficient, the 1 mM NADH might be too little to see product formation or only traces. However, I think in your setup you should have seen a little bit of product – so not point to repeat. ****** -->6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .--> Reviewer #1: No Reviewer #2: Yes: Frank Hollmann Reviewer #3: Yes: Dirk Tischler ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.--> https://doi.org/10.1371/journal.pone.0332702.r001
Revision 1
20 Jul 2025 Author Response Review Comments to the Author Reviewer #1 This study reports on something that is often encountered, but is rarely published – a negative result. In this case, a fairly exhaustive attempt was made to replicate the prior, published observation that LadA has n-alkane monooxygenase activity in vitro. An array of assay conditions were attempted and multiple LadA homologs from other organisms were assessed. In all cases, no n-alkane monooxygenase activity was detected, suggesting that LadA-like enzymes are unable to catalyze the previously published reaction and the true function of LadA is still unknown. The fact that this was independently evaluated by two separate groups in this study (Noda-Garcia and Teufel groups) provides an additional level of confidence in the findings. This study appears to be well-conducted and provides information that will be useful for anyone studying LadA. However, I have one major suggesting for the authors before publishing this study: 1. Assess LadA for activity under conditions with MgSO4, with and without Fre, similar to the published studies, even though they do not seem mechanistically sound. I realize that there is no rationale for needing Mg2+ and a flavin reductase is usually necessary for these two component systems to function. In my experience, NADH can nonenzymatically reduce FMN, albeit poorly, so it is conceivable that FMNH2 could still be generated in the assay without Fre. I appreciate that the results of this additional experiment will very likely be the same as all of your others (no activity). Without it, though, a skeptical reader of your study may wonder if you didn’t observe activity with LadA simply because you opted not to use similar conditions to the prior studies. Thank you for your comment. Indeed, the absence of identical conditions might raise questions. We performed the experiments as originally reported by adding Mg2+ with and without Fre. As expected, we did not observe any hexadecane conversion or hexadecanol formation. These results are now explicitly referenced in the main text, and the chromatograms are presented as Supplementary Figure 1. Also, due to this new experiment the test now read as: “In the study by Feng et al. (2007), the reaction setup included 1 mM hexadecane and 1 mM MgSO₄ in a 50 mM Tris-HCl buffer at pH 7.5, along with 1 mM NADH. It is unclear how LadA activity could be detected in those experiments without FMN added, since Group C FPMOs usually do not purify with FMN bound. The second study by Li et al. (2008), which focused on the structural analysis of LadA, also used 1 mM hexadecane in a 50 mM Tris-HCl buffer at pH 7.5; however, NADH was omitted, and 1 mM FMNH2 was added instead. No information was provided to describe how the FMNH2 was generated for these experiments. This is critical information as it is unstable under aerobic conditions. ” Minor suggestion: 2. Be a little more forceful in pointing out how nonsensical some of the conditions are from the prior published studies. “In the study by Feng et al. (2007), the reaction setup included 1 mM hexadecane and 1 mM MgSO₄ in a 50 mM Tris-HCl buffer at pH 7.5, along with 1 mM NADH.” It is unclear how LadA activity could be detected in those experiments without FMN added since Group C FMOs usually do not purify with FMN bound. “The second study by Li et al. (2008), which focused on the structural analysis of LadA, also used 1 mM hexadecane in a 50 mM Tris-HCl buffer at pH 7.5; however, NADH was omitted, and 1 mM FMNH2 was added instead.” No information was provided to describe how the FMNH2 was generated for these experiments. This is critical information, as FMNH2 is not stable under aerobic conditions. Thanks for the observations. The text was corrected, and the three new sentences suggested were added. Reviewer #2 This manuscript addresses the challenging issue of n-alkane functionalization. The authors have undertaken an effort to clarify the enzyme activity of the flavin-dependent enzyme LadA, an enzyme previously suggested to function as a long-chain n-alkane monooxygenase. Their rigorous experimentation, including testing optimized variants and novel homologs under in vitro conditions, significantly advances the current understanding. Our research group has also been particularly intrigued by LadA and pursued extensive studies aimed at reproducing the previously reported literature findings, unfortunately without success. Therefore, we highly appreciate the authors' systematic approach in clearly demonstrating and openly discussing the reproducibility challenges and limitations related to LadA activity. Their results provide crucial clarity and offer a balanced and critical evaluation of the previously reported roles of LadA and related flavoprotein monooxygenases. I congratulate the authors on this valuable and rigorous study, which strongly contributes to redefining the biochemical understanding of these enzymes. Their findings represent an essential milestone and open new avenues for research to uncover the true biochemical roles of LadA and its homologs. Thank you for your kind words. We agree that our attempts in investigating LadA’s activity may ultimately contribute to uncovering its native function and advance the broader understanding of FPMOs and enzymatic C–H functionalization. Reviewer #3 Dear Authors that is a very important finding and clarifies many open questions we had for this special FPMO, it also demonstrates how experimental setup can be designed to answer research questions from different perspectives. I have some minor mostly text-related suggestions which I hope help to improve the manuscript. Thank you very much for your thoughtful feedback. We're glad to hear that the findings help clarify open questions regarding this unusual FPMO. We fully agree that the design of the experimental setup was key to approaching the problem from multiple angles. We appreciate your text-related suggestions and will incorporate them to further improve the clarity and quality of the manuscript. Comments: - „sp.“ should read italics (e.g., page 3 “AlmA in Acinetobacter …” Corrected. - “E. coli” should read italics Corrected. - “(18 μM) LadA to our assays,,” comma issue to be corrected Corrected. - Be consistent with “n-alkane” in terms of “n” reads italics Corrected. “n-alkane” is consistent throughout the text. - Protein expression is wrong; only genes can be expressed; correct throughout. Corrected. The wording ”protein expression” and variants were changed to “protein production” throughout the text. - In your assays you provide Fre to provide reduced flavin for the potential monooxygenase; did you observe any H2O2 formation and if; was a different plus/minus substrate being present, that would at least indicate a ligand binding and thus effector role. We did not measure H2O2 formation. - Fre might not be the best reductase partner as some two-component FPMOs need a certain partner to become active; like cooperativity and this might be an important conclusion to search for the natural redox couple. This is an interesting observation. Our decision to use a previously characterized flavin reductase was based on the fact that ladA is most likely acquired through horizontal gene transfer—being encoded on a plasmid in the native host—while no flavin reductase gene is present on the same plasmid. This supports the hypothesis that LadA may interact promiscuously with host-encoded flavin reductases. Group C FPMOs have generally been shown to function promiscuously with reductase partners. See references Su, T., 2018: https://doi.org/10.3389/fmicb.2018.00231 Matthews A., 2020: https://doi.org/10.1038/s41589-020-0476-2 Mathews, A., et al., 2021: https://doi.org/10.1111/febs.16193 Moreover, the transfer of reduced flavin from the reductase to the monooxygenase component has primarily been proposed to occur via free diffusion. A comprehensive review on this topic is provided by Sucharitakul et al. (2014) https://doi.org/10.1016/j.abb.2014.05.009. - If the potential uncoupling was high and the redFMN transfer not efficient, the 1 mM NADH might be too little to see product formation or only traces. However, I think in your setup you should have seen a little bit of product – so not point to repeat. Thank you. Indeed, we hypothesized that an excess of NADH would be sufficient to observe at least trace amounts of hexadecane conversion. Attachments Attachment Submitted filename: response_to_reviewers.docx https://doi.org/10.1371/journal.pone.0332702.r002
4 Sep 2025 Decision Letter - Paul Aurelian Gagniuc, Editor Can flavoprotein monooxygenases functionalize long-chain n-alkanes? PONE-D-25-27398R1 Dear Dr. Noda-Garcia, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. 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Has the statistical analysis been performed appropriately and rigorously? --> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** -->4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.--> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** -->5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.--> Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** -->6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)--> Reviewer #1: (No Response) Reviewer #2: A very important paper. Ich have no further objections and I am a bit disturbed that my answer needs to be at least 100 characters. Reviewer #3: All previous comments have been addressed. Well done! No further changes are needed from point of view. ****** -->7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .--> Reviewer #1: No Reviewer #2: Yes: Frank Hollmann Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0332702.r003
Formally Accepted
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