Investigating the native functions of [NiFe]-CODH through genomic context analysis
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eLife Assessment
This valuable work analyzes a large dataset of [NiFe]-CODHs, integrating genomic context, operon organization, and clade-specific gene neighborhoods to discern patterns of diversification and adaptation. A consistent examination of CODH genomic contexts, including CODH-HCP co-occurrence, informs interpretations of enzymatic activity, biotechnological potential, and differential functional roles, in line with current standards in genomic enzymology. With solid support, this work provides a broadly informative contribution to the field.
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Abstract
Carbon monoxide dehydrogenases containing nickel-iron active sites ([NiFe]-CODHs) catalyze the reversible oxidation of CO to CO2, representing key targets for biocatalytic CO2 reduction. Despite dramatic differences in catalytic rates and O2 tolerance between CODH variants, the molecular basis for this functional diversity remains poorly understood. We applied comparative genomics and synteny analysis to investigate the biochemical roles of CODH clades A-F using 1,376 CODH and 1,545 hybrid cluster protein sequences. Around 30% of genomes encode multiple CODH isoforms. Analysis revealed distinct gene clustering patterns correlating with biochemical function. Clades A, E, and F exhibit a degree of distributional exclusivity. Clades C and D frequently co-occur with active CODHs, suggesting auxiliary roles. Operon architecture analysis revealed functional specialization: clade A links to acetyl-CoA synthase; clades A, E and F contain essential maturation machinery (CooC, CooJ, CooT) correlating with catalytic activity; clade B associates with transporters; clade C with electron transfer partners; clade D with transcriptional regulators. High CODH-HCP co-occurrence (except clade A) suggests functional or environmental interdependency. These findings establish clades A, E and F as primary biocatalyst targets while defining regulatory functions for clades C and D, providing a genomics framework for predicting CODH phenotypes.
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eLife Assessment
This valuable work analyzes a large dataset of [NiFe]-CODHs, integrating genomic context, operon organization, and clade-specific gene neighborhoods to discern patterns of diversification and adaptation. A consistent examination of CODH genomic contexts, including CODH-HCP co-occurrence, informs interpretations of enzymatic activity, biotechnological potential, and differential functional roles, in line with current standards in genomic enzymology. With solid support, this work provides a broadly informative contribution to the field.
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Reviewer #1 (Public review):
Summary:
This manuscript analyzes a large dataset of [NiFe]-CODHs with a focus on genomic context and operon organization. Beyond earlier phylogenetic and biochemical studies, it addresses CODH-HCP co-occurrence, clade-specific gene neighborhoods, and operon-level variation, offering new perspectives on functional diversification and adaptation.
Strengths:
The study has a valuable approach.
Comments on revised version:
I am satisfied that the authors have adequately addressed my previous comments in the revised manuscript.
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Reviewer #2 (Public review):
The authors present a comparative genomic and phylogenetic analysis aimed at elucidating the functions of nickel-dependent carbon monoxide dehydrogenases (Ni-CODHs) and hybrid-cluster proteins (HCPs). By examining gene neighborhoods, phylogenetic relationships, and co-occurrence patterns, they propose functional hypotheses for different CODH clades and highlight those with the greatest potential for biotechnological applications.
A major strength of this work lies in its systematic and conceptually clear approach, which provides a rapid and low-cost framework for predicting the functional potential of newly identified CODHs based on sequence data and genomic context. The analysis is careful in minimizing false positives and offers valuable insights into the diversity and distribution of CODH enzyme clades.
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Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
(1) Rationale for excluding clades G and H and clarification of clade definitions
We appreciate this important request for clarification. In the revised manuscript, we now explicitly state (Methods, Tree generation) that the phylogenetic framework used in this study follows the clade definitions established by Techtmann et al. (Front. Microbiol. 2012, 3, 132), which classify [NiFe]-CODHs into clades based on high supporting values in nodes (bootstrap >75). We deem Techtmann et al.’s work as best lead, since their approach with two different types of trees (ML vs. Bayesian) gives solid support to this classification of clades. We ourselves did not perform Bayesian statistics, instead we used the known clades …
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
(1) Rationale for excluding clades G and H and clarification of clade definitions
We appreciate this important request for clarification. In the revised manuscript, we now explicitly state (Methods, Tree generation) that the phylogenetic framework used in this study follows the clade definitions established by Techtmann et al. (Front. Microbiol. 2012, 3, 132), which classify [NiFe]-CODHs into clades based on high supporting values in nodes (bootstrap >75). We deem Techtmann et al.’s work as best lead, since their approach with two different types of trees (ML vs. Bayesian) gives solid support to this classification of clades. We ourselves did not perform Bayesian statistics, instead we used the known clades from literature to assign ours.
Clades G and H were not deliberately excluded from downstream genomic-context and operon analyses. They were excluded by our pipeline, because their data did not fulfil our initial quality assessments, such as: host classified down to species level (https://github.com/boehmax/protein-per-organism), and protein exists in the IPG database of NCBI (https://github.com/boehmax/protein-to-genome).
Clade G and H are both represented by only a very small number of sequences, most of which derive from fragmented or poorly annotated genomes, preventing reliable assessment of operon organization and gene neighbourhood conservation. As a result, inclusion of these clades would not allow statistically meaningful or biologically interpretable comparisons with clades A–F.
To improve transparency, we have added a brief explanation of these limitations in the Results (Results, Neighbor analysis).
(2) Presentation and interpretation of co-occurrence data
We agree that the presentation of the co-occurrence data required improvement. In the revised supplementary material, we now include a table in the long format that might be easier to interpret than a matrix representation as seen in Fig. 3B.
We have also revised the Results text to more precisely reflect the numerical trends. Specifically, we clarify that clade D shows co-occurrence with clades A, E and F, while clade C only displays co-occurrence with clade E. The statement that clades C and D “more often co-occur” has been removed and rephrased to avoid overgeneralization and to better align with the quantitative data shown in Figure 3B and the supplementary table (Results, Co-occurrence and Correlation).
(3) Rationale for operon-level rather than organism-level analysis
We thank the reviewer for highlighting this conceptual point. In the revised manuscript, we now explicitly state that our analysis was conducted at the operon level because individual genomes frequently encode multiple CODH operons that are phylogenetically and functionally distinct. Treating each operon as an independent functional unit allows us to capture this intra-genomic diversity and to associate specific gene neighbourhoods with individual CODH clades. We furthermore discuss in the introduction explicitly technical reasons why we decided to limit this study to the operon level for more transparency.
Nevertheless, we acknowledge that this approach may overlook higher-level regulatory or physiological interactions among multiple CODHs encoded within the same genome. This limitation is now discussed explicitly, and we acknowledge that operon-level analysis should be a complementary, not exhaustive, framework for functional inference.
Reviewer #2 (Public review):
We thank Reviewer #2 for their positive assessment of the conceptual clarity and methodological utility of our approach, as well as for their thoughtful discussion of its limitations.
Regarding incomplete genome assemblies, limited representation of class II HCPs, and potential omission of distal pathway components, we agree fully. We stress that our conclusions are probabilistic and hypothesis-generating rather than definitive functional assignments.
In response to the concern about reproducibility of the visual filtering step, we have added a more explicit description (Methods, Data collection and refinement) of the criteria used to exclude non-CODH homologs (e.g., absence of conserved active-site motifs, unknown folds predicted with AlphaFold3, extremely long tree branches). This clarification improves transparency and facilitates replication of the analysis.
Finally, we concur that extrapolating enzymatic activity or inactivity from a limited number of characterized representatives should be done cautiously. We have revised the wording throughout the manuscript to further temper such generalizations and to frame our interpretations explicitly as predictions that require experimental validation.
Once again, we thank both reviewers for their constructive feedback, which has significantly improved the clarity, rigor, and transparency of the manuscript. We believe that the revisions address all concerns raised and strengthen the overall contribution of this work.
Recommendation from authors:
Reviewer #1 (Recommendations for the authors):
All suggested editorial and stylistic corrections were implemented. These include refinements to the wording in the Abstract, grammatical corrections, streamlined phrasing, standardized figure callouts and supplementary file references, corrected abbreviations, and consistent formatting of references and author names. The only exception concerns the suggested change from MetCODH to MtCODH. We have retained MetCODH, as this abbreviation is well established in the literature for the Methanothermobacter thermophila CODH and is commonly used in prior studies (e.g., https://doi.org/10.1073/pnas.2410995121 ). MtCODH has historically been referring to CODH from Neomoorella thermoacetica (previously Moorella thermoacetica, hence the abbreviation Mt). We chose to rename that to NtCODH but to avoid confusion, keep MetCODH for Methanothermobacter thermophila.
Reviewer #2 (Recommendations for the authors):
We likewise addressed the majority of recommendations. We now report the versions of all software tools and databases used, standardized capitalization and naming of software and platforms (e.g., GitHub, eggNOG), clarified the BLAST implementation and database employed, and added direct repository links for custom scripts in both the Methods section and the bibliography. Overall grammatical consistency and formatting were improved throughout the manuscript. In addition, the criteria and procedure used for visual inspection to remove non-CODH sequences are now described more explicitly to enhance reproducibility, and several methodological sections were streamlined as suggested. Minor textual redundancies were removed, and phrasing was simplified where appropriate.
Figure legends and formatting were revised to improve clarity and consistency. Adjustments to color usage and font consistency were made where feasible to enhance readability. The color scheme in Figure 1 was adjusted as suggested, and darker shades were chosen for clade H and G. This change was also implemented in the Supplementary File 9_Tree5. Figure 3A was retained, as it provides information on the frequency of multiple CODHs from the same clade within genomes, which cannot be inferred from the probability matrix shown in Figure 3B; together, these panels offer complementary insights. We adjusted the figure caption to make this clearer. We increased the visibility of data points in Figure 4B. To allow inclusion of the full dataset we did not collapse the x-axis as suggested. Figure 4C was retained in its original format to emphasize the characteristic operon “fingerprints” of each CODH clade, which is a central focus of this work. A table is supplied in Supplementary File 2, which allows data exploration with the preferred focus of the reader.
A small number of suggestions were therefore not implemented exactly as proposed, primarily where alternative revisions were judged to better preserve clarity or analytical intent. These decisions are minor and do not affect the conclusions or reproducibility of the study.
Overall, we believe that these revisions have substantially improved the manuscript’s readability, transparency, and technical rigor, and we thank the reviewers again for their careful and constructive feedback.
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eLife Assessment
This study presents a valuable analysis of a large dataset of [NiFe]-CODHs, integrating genomic context, operon organization, and clade-specific gene neighborhoods to discern patterns of functional diversification and adaptation. Carefully looking at the CODH genomic context, e.g., CODH-HCP co-occurrence, the authors gain insight into enzymatic activity, biotechnological potential, and differential functional roles. The approach aligns with current standards in genomic enzymology to characterize newly identified enzymes. With solid support, this work provides a broadly informative contribution to the field.
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Reviewer #1 (Public review):
Summary:
This manuscript analyzes a large dataset of [NiFe]-CODHs with a focus on genomic context and operon organization. Beyond earlier phylogenetic and biochemical studies, it addresses CODH-HCP co-occurrence, clade-specific gene neighborhoods, and operon-level variation, offering new perspectives on functional diversification and adaptation.
Strengths:
The study has a valuable approach.
Weaknesses:
Several points should be addressed.
(1) The rationale for excluding clades G and H should be clarified. Inoue et al. (Extremophiles 26:9, 2022) defined [NiFe]-CODH phylogenetic clades A-H. In the present manuscript, clades A-H are depicted, yet the analyses and discussion focus only on clades A-F. If clades G and H were deliberately excluded (e.g., due to limited sequence data or lack of biochemical evidence), …
Reviewer #1 (Public review):
Summary:
This manuscript analyzes a large dataset of [NiFe]-CODHs with a focus on genomic context and operon organization. Beyond earlier phylogenetic and biochemical studies, it addresses CODH-HCP co-occurrence, clade-specific gene neighborhoods, and operon-level variation, offering new perspectives on functional diversification and adaptation.
Strengths:
The study has a valuable approach.
Weaknesses:
Several points should be addressed.
(1) The rationale for excluding clades G and H should be clarified. Inoue et al. (Extremophiles 26:9, 2022) defined [NiFe]-CODH phylogenetic clades A-H. In the present manuscript, clades A-H are depicted, yet the analyses and discussion focus only on clades A-F. If clades G and H were deliberately excluded (e.g., due to limited sequence data or lack of biochemical evidence), the rationale should be clearly stated. Providing even a brief explanation of their status or the reason for omission would help readers understand the scope and limitations of the study. In addition, although Figure 1 shows clades A-H and cites Inoue et al. (2022), the manuscript does not explicitly state how these clades are defined. An explicit acknowledgement of the clade framework would improve clarity and ensure that readers fully understand the basis for subsequent analyses.
(2) The co-occurrence data would benefit from clearer presentation in the supplementary material. At present, the supplementary data largely consist of raw values, making interpretation difficult. For example, in Figure 3b, the co-occurrence frequencies are hard to reconcile with the text: clade A shows no co-occurrence with clade B and even lower tendencies than clades E or F, while clade E appears relatively high. Similarly, the claim that clades C and D "more often co-occur, especially with A, E, and F" does not align with the numerical trends, where D and E show stronger co-occurrence but C does not. A concise, well-organized summary table would greatly improve clarity and prevent such misunderstandings.
(3) The rationale for analyzing gene neighborhoods at the single-operon level needs clarification. Many microorganisms encode more than one CODH operon, yet the analysis was carried out at the level of individual operons. The authors should clarify the biological rationale for this choice and discuss how focusing on single operons rather than considering the full complement per organism might affect the interpretation of genomic context.
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Reviewer #2 (Public review):
The authors present a comparative genomic and phylogenetic analysis aimed at elucidating the functions of nickel-dependent carbon monoxide dehydrogenases (Ni-CODHs) and hybrid-cluster proteins (HCPs). By examining gene neighborhoods, phylogenetic relationships, and co-occurrence patterns, they propose functional hypotheses for different CODH clades and highlight those with the greatest potential for biotechnological applications.
A major strength of this work lies in its systematic and conceptually clear approach, which provides a rapid and low-cost framework for predicting the functional potential of newly identified CODHs based on sequence data and genomic context. The analysis is careful in minimizing false positives and offers valuable insights into the diversity and distribution of CODH enzyme clades.
Ho…
Reviewer #2 (Public review):
The authors present a comparative genomic and phylogenetic analysis aimed at elucidating the functions of nickel-dependent carbon monoxide dehydrogenases (Ni-CODHs) and hybrid-cluster proteins (HCPs). By examining gene neighborhoods, phylogenetic relationships, and co-occurrence patterns, they propose functional hypotheses for different CODH clades and highlight those with the greatest potential for biotechnological applications.
A major strength of this work lies in its systematic and conceptually clear approach, which provides a rapid and low-cost framework for predicting the functional potential of newly identified CODHs based on sequence data and genomic context. The analysis is careful in minimizing false positives and offers valuable insights into the diversity and distribution of CODH enzyme clades.
However, several limitations should be considered when interpreting the findings. The use of incomplete genome assemblies may lead to the exclusion of relevant genes or operonic regions. Clade H was omitted due to a lack of information on its host, and the number of class II HCPs included is limited. Although the genomic window analyzed is relatively broad, it may still miss functionally relevant neighboring genes. The study assumes that the pathways associated with CODHs are encoded near the enzyme loci, but these could also occur elsewhere in the genome or on the complementary strand. The authors acknowledge these and other limitations clearly and thoughtfully, which strengthens the transparency and credibility of their analysis.
Given the high evolutionary diversity of CODHs-both across and within clades-phenotypic predictions derived solely from sequence and neighborhood data should be interpreted with caution. Sequence-based searches, while specific, may have limited sensitivity, and structural homology searches could further enrich the dataset. Additionally, the visual inspection used to filter out non-CODH sequences is not described in detail, leaving uncertainty about reproducibility. The generalization of enzymatic activity or inactivity from a few characterized examples to entire clades should also be regarded as tentative.
Despite these limitations, the study presents a solid and valuable methodological framework that can aid in the rapid functional screening of novel CODH enzymes and may inspire broader applications in enzyme discovery and metabolic annotation. -
