Nitrogenase resurrection and the evolution of a singular enzymatic mechanism
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eLife assessment
This manuscript reports valuable findings regarding the evolution of nitrogenases through ancestral sequence reconstruction and resurrection. The results are solid and support the conclusions of the study, and highlight the historical constraints that have been acting on this enzyme. The findings will be of interest for people interested in enzyme evolution in general and particularly for those interested in the evolution of nitrogenases.
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Abstract
The planetary biosphere is powered by a suite of key metabolic innovations that emerged early in the history of life. However, it is unknown whether life has always followed the same set of strategies for performing these critical tasks. Today, microbes access atmospheric sources of bioessential nitrogen through the activities of just one family of enzymes, nitrogenases. Here, we show that the only dinitrogen reduction mechanism known to date is an ancient feature conserved from nitrogenase ancestors. We designed a paleomolecular engineering approach wherein ancestral nitrogenase genes were phylogenetically reconstructed and inserted into the genome of the diazotrophic bacterial model, Azotobacter vinelandii, enabling an integrated assessment of both in vivo functionality and purified nitrogenase biochemistry. Nitrogenase ancestors are active and robust to variable incorporation of one or more ancestral protein subunits. Further, we find that all ancestors exhibit the reversible enzymatic mechanism for dinitrogen reduction, specifically evidenced by hydrogen inhibition, which is also exhibited by extant A. vinelandii nitrogenase isozymes. Our results suggest that life may have been constrained in its sampling of protein sequence space to catalyze one of the most energetically challenging biochemical reactions in nature. The experimental framework established here is essential for probing how nitrogenase functionality has been shaped within a dynamic, cellular context to sustain a globally consequential metabolism.
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Author Response
Reviewer #1 (Public Review):
Understanding the evolution of nitrogenases is a very important problem in the field of evolutionary biogeochemistry. Ancestral sequence reconstruction at least in theory could offer insights into how this planet alerting activity evolved from ancestors that did not reduce nitrogen. But the very many components of the nitrogenase enzyme system make this a very challenging question to answer.
This paper now demonstrates the first empirical resurrection of functional ancestral nitrogenases both in vivo and in vitro. The nodes that are resurrected are very shallow in the nitrogenase tree and do not help answer how these proteins evolved. The authors' reasoning for choosing these nodes is that they are likely compatible with the metal cluster assembly machinery of their chosen host organism, …
Author Response
Reviewer #1 (Public Review):
Understanding the evolution of nitrogenases is a very important problem in the field of evolutionary biogeochemistry. Ancestral sequence reconstruction at least in theory could offer insights into how this planet alerting activity evolved from ancestors that did not reduce nitrogen. But the very many components of the nitrogenase enzyme system make this a very challenging question to answer.
This paper now demonstrates the first empirical resurrection of functional ancestral nitrogenases both in vivo and in vitro. The nodes that are resurrected are very shallow in the nitrogenase tree and do not help answer how these proteins evolved. The authors' reasoning for choosing these nodes is that they are likely compatible with the metal cluster assembly machinery of their chosen host organism, A. vinelandii. The reader is left to wonder if deeper, more interesting nodes were tried but didn't yield any activity. As the paper stands, it proves that relatively shallow nitrogenase ancestors can be resurrected, but these nodes do not yet teach us anything very fundamental about how these enzymes evolved.
Technically, this work was no doubt challenging. Genome engineering in A vinelandii is very difficult and time-consuming. This organism was chosen because it is an obligate aerobe, which makes it easier to handle than the many anaerobic bacteria and archaea that harbor nitrogenases. It does make one wonder if this choice of organism is wise: the authors themselves note that it probably has a set of specialized proteins that allow the nitrogenase to be assembled and function in the presence of oxygen. This may limit A. vinelandii's potential future ancestral reconstructions deeper in the tree, which according to the authors' reasoning probably requires different assembly machinery.
The ancestral sequence reconstruction is done in two different ways: Two out of three reconstructions are carried out with what appears to be an incorrect algorithm implemented in older versions of RaxML. This algorithm is not a full marginal reconstruction, because it only considers the descendants of the node of interest for the reconstruction. The full algorithm (implemented e.g. in PAML and the newest versions of RaxML) considers all tips for a marginal reconstruction. The fact that this was called a marginal ancestral sequence reconstruction in RaxML's manual is unfortunate - as far as I understand it is in fact just the internal labelling of nodes produced by the pruning algorithm, which is not equivalent to a marginal reconstruction. In this specific case, it is unlikely that this has led to any fundamental issues with the reconstructions (as all are functional nitrogenases, which is to be expected in this part of the tree). For the shallower of the two nodes, the authors in fact verify that they get the same experimental results if they use PAML's full implementation of a marginal reconstruction (which yields a somewhat different sequence for this node). It would have been helpful to point this RaxML-related issue out in the methods, so as to prevent others from using this incorrect implementation of the ASR algorithm.
One other slightly confusing aspect of the paper is that it contains two different maximum likelihood trees, which were apparently inferred using the same dataset, model, and version of RaxML. It is unclear why they have different topologies. This probably indicates a lack of convergence. Again, this does not cast any doubt on the uncontroversial findings of this paper that shallow nodes within the nitrogenases are also nitrogenases.
We thank the reviewer for their careful appraisal of our article, and their helpful recommendations for improving its quality. We appreciate the reviewer’s comment regarding the experimental challenges associated with nitrogenase engineering and genetic studies of our bacterial model, Azotobacter vinelandii. The complexity of nitrogen fixation machinery does indeed present several experimental obstacles, though, as we note in our revised article, this feature also makes the systems-level approach we have implemented here ideal for evolutionary studies of nitrogenases and their associated network.
The reviewer focuses on three central points: 1) the relevance of the targeted ancestral nodes for addressing fundamental questions concerning nitrogenase origins, 2) the applicability of our bacterial model for older reconstructions, and 3) issues associated with the different trees/methods for ancestral sequence reconstruction.
Addressing the first point, we concede that targeting relatively shallow nodes cannot specifically test hypotheses concerning the earliest stages of nitrogenase evolution (e.g., “how this planet altering activity evolved from ancestors that did not reduce nitrogen”). Our central result is that a specific, enzymatic mechanism for dinitrogen binding reduction (established for three modern nitrogenases to date) extends back through nitrogenase ancestry over the studied timeline. More broadly, a conserved nitrogenase mechanism in the only surviving family of nitrogenase families suggests that life may have been constrained in its available strategies for achieving this challenging biochemical reaction. By comparison, multiple abiotic pathways for nitrogen fixation are feasible, and another, ecologically vital metabolism, carbon fixation, can proceed by at least seven pathways. Deeper investigations into these possible evolutionary constraints and across deeper portions of the nitrogenase tree will require continued study, which we anticipate will be facilitated by the experimental approach presented in this article.
Concerning the applicability of our bacterial model, we agree that it is possible that older reconstructions may require different host organisms so as to provide a compatible genetic background. Similar considerations we have outlined in our article, including a systematic evaluation of the genetic components that likely accompanied nitrogenase ancestors in their ancient hosts, will likely be necessary. Nevertheless, we foresee that the general, systems-level approach that we have built for Azotobacter can be adapted for additional microbial models, and that these efforts will be worthwhile given the significance of biological nitrogen fixation to evolutionary biogeochemistry and microbial engineering applications.
Finally, we thank the reviewer for noting the differences in the ancestral sequence reconstruction algorithms of RAxML v.8 and PAML and welcome an explanation of these issues in our revised article. We confirm that RAxML v.8 does not perform full marginal reconstruction (in contradiction to its description in the RAxML manual). Due to this concern, we repeated our ancestral sequence reconstruction with PAML, which, like newer versions of RAxML, does implement the full algorithm. Here, ancestors reconstructed by RAxML v.8 and PAML from equivalent phylogenetic nodes yield comparable experimental results, indicating that the algorithm differences have not significantly impacted the major outcomes of our study. In the second analysis, we repeated the entire phylogenetic ancestral sequence reconstruction workflow, though did not trim the alignment as we did in the first case (this has now been clarified). This likely explains the differences in our trees, as the reviewer notes. We have included these details in the Materials and Methods section of our revised article.
In addition to expanding upon the points outlined above throughout the revised article, we have included additional text in the Discussion that elaborates on the limitations of our study, and in particular, the need to explore deeper portions of the nitrogenase tree in future work.
Reviewer #2 (Public Review):
The authors convincingly show that their reconstructed ancestral nitrogenases are active both in vivo and in vitro, and show similar inhibitory effects as extant/wild-type enzymes.
The conclusion that, evolutionarily, there is a "single available mechanism for dinitrogen reduction" is not well explored in the paper. This suggests a limitation of using ancestral sequence reconstruction in this instance.
We thank the reviewer for their comments and appreciate their assessment that the core experimental results are conclusively demonstrated, including in vivo/in vitro activity of ancestral nitrogenase enzymes and that they all exhibit the specific mechanism for dinitrogen binding and reduction, evidenced by hydrogen inhibition.
We note the reviewer’s concern regarding the evolution of the dinitrogen reduction mechanism described above. Our primary conclusion is that this mechanism is conserved in the studied nitrogenase ancestors, which, together with previous demonstrations of this mechanism in the different nitrogenase isozymes (Mo, V, Fe) of Azotobacter vinelandii, suggests that this is an early evolved feature of the nitrogenase family. These enzymes have thus not only been performing an ecologically vital, metabolic function, but have likely been achieving this challenging biochemical reaction in the same manner for billions of years. We discuss the resulting implications as they relate to evolutionary constraints on biological nitrogen fixation strategies. We clarify that our presented paleomolecular approach cannot directly evaluate alternate evolutionary scenarios that did not persist and were not preserved in extant genomic sequences, as ancestral sequence reconstruction is fundamentally informed by extant sequence diversity. Our approach is a powerful tool for defining the contours of ancestral nitrogenase sequence-function space, which can serve as a basis for engineering and evaluating alternate scenarios. We have clarified these points in our Discussion.
Reviewer #3 (Public Review):
In this work, the authors attempt to probe the constraints on the early evolution of nitrogen fixation, the development of which presented a key metabolic transition. Given that life on Earth evolved only once (to our knowledge) which aspects were necessary and which may have taken a different course are open questions. Are there alternative forms of life, metabolic networks, or even enzymatic mechanisms that could have replaced the ones we see today, or is the space of possible biologies limited? This manuscript tests the ability of ancestrally-reconstructed molybdenum-dependent nitrogenase complexes to support diazotrophic growth in Azotobacter vinelandii, as well as in vivo and in vitro activity, which all point towards a conserved mechanism for nitrogen reduction at least since proteobacteria divergence.
This is an ambitious project, requiring multiple techniques, systems, and approaches, and the successful combination of these is one of the major strengths of this work. Using parallel techniques is an important way to be certain that the overall results are robust, and an appropriate mix of in vivo and in vitro experiments is chosen here. The manuscript should serve as a useful model for how to combine phylogenetics and biochemistry.
The nature of ASR means that a solid phylogeny and/or understanding of how robust the results are to uncertainty in reconstructed states is essential since all results flow from there. The overall phylogenetic methods used are appropriate and the system is an apt one for the technique, but there is not quite enough detail in the methods to be certain of the results. Given that only the single maximum a posteriori sequence is assayed at every 3 nodes, this may have compounding results in that the sensitivity to uncertainty in the reconstruction is increased. The authors appropriately make qualitative rather than quantitative inferences, but some hesitation towards the overall results still exists.
The assumption that the Anc1A/B and Anc2 nodes correspond to ancestral states might be undermined by horizontal gene transmission, which has been reported for nif clusters. In particular, there may be different patterns of transmission for each element of the cluster. By performing reconstruction with a concatenated alignment, the phylogenetic signal is potentially maximized, but with the assumption that each gene has an identical history. Discordant transmission may cause an incorrect topology to be recovered.
Finally, I am unsure if ASR is the most appropriate approach to answer questions of contingency and alternative pathways for protein evolution. ASR may tell what nitrogenase millions or billions of years ago looked like, but it can only say what has already existed. If there are different mechanisms or metabolic pathways enabling nitrogen fixation that simply never came to pass, via contingency and entrenchment or simple chance, ASR would say nothing about them. It is true that a conserved mechanism would point towards a constrained space for evolving nitrogen fixation, but that does not directly address it.
Overall, despite these issues, the manuscript is compellingly written and the figures are attractive and clear, and help get the major narrative across. This work will be of interest to protein biochemists of evolutionary bent and microbial physiologists with an interest in the origins of life.
We thank the reviewer for their evaluation of our study and appreciate their comments regarding the experimental effort involved and scientific significance. We have carefully considered their recommendations to improve our article.
The reviewer’s critical comments concern 1) the level of detail regarding the phylogenetic methodology, 2) the impact of horizontal gene transfer on phylogenetic reconstructions, and 3) the appropriateness of ancestral sequence reconstruction for accessing alternate evolutionary scenarios in the emergence of biological nitrogen fixation.
We have addressed the first point by including additional methodological details regarding our phylogenetic analyses in our Materials and Methods section, including alignment and model testing tools, as well as our rationale for using two ancestral sequence reconstruction methods, RAxML and PAML.
Regarding the second point, we acknowledge that horizontal gene transfer has played a significant role in the evolution and distribution of biological nitrogen fixation, which has been established and explored in previous work by others. We have included in our Discussion an additional paragraph which addresses potential impact of horizontal gene transfer in nitrogenase evolution. Though we do not expect horizontal transfer to contribute a significant source of uncertainty in the timeline studied for the reasons discussed in the revised manuscript, we agree that it is an important consideration for future work and that may impact reconstructions in other lineages within the nitrogenase phylogeny.
Finally, in new text within the Discussion, we also acknowledge that ancestral sequence reconstruction cannot yet directly test alternate historical scenarios. We have clarified our language concerning conservation and constraints in the evolution of biological nitrogen fixation. Because ancestral sequence reconstruction is informed by modern sequences, it is limited to exploring the historical sequence space within their shared ancestry. It is therefore possible that, early in the history of life, there were multiple enzymatic strategies for fixing nitrogen, and that they were outcompeted and thus have left no trace in modern genomes. Another possibility is that these alternate strategies simply never evolved.
In the present study, we have identified a pattern of conservation with regard to a specific mechanism for dinitrogen binding and reduction, suggesting a level of evolutionary constraint that can be further interrogated. For example, ancestral sequence reconstruction, as implemented in our nitrogenase resurrection strategy, can be used to empirically investigate the underlying sources of these constraints. We note that despite decades of research in this domain, a full understanding of how nitrogenases perform this remarkable metabolic step, both today and in the past, remains elusive (as other reviewers of the present study have also noted). Evolutionarily informed studies of nitrogenase function enabled by ASR can reveal the design principles that have shaped its direct ancestry, which can potentially serve as a basis for engineering alternative molecular strategies for nitrogen fixation. The power of the molecular paleogenetic approach here is in extending functional investigations beyond the sequence space occupied by modern nitrogenase and identifying patterns in their functional variation through their evolutionary histories.
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eLife assessment
This manuscript reports valuable findings regarding the evolution of nitrogenases through ancestral sequence reconstruction and resurrection. The results are solid and support the conclusions of the study, and highlight the historical constraints that have been acting on this enzyme. The findings will be of interest for people interested in enzyme evolution in general and particularly for those interested in the evolution of nitrogenases.
-
Reviewer #1 (Public Review):
Understanding the evolution of nitrogenases is a very important problem in the field of evolutionary biogeochemistry. Ancestral sequence reconstruction at least in theory could offer insights into how this planet alerting activity evolved from ancestors that did not reduce nitrogen. But the very many components of the nitrogenase enzyme system make this a very challenging question to answer.
This paper now demonstrates the first empirical resurrection of functional ancestral nitrogenases both in vivo and in vitro. The nodes that are resurrected are very shallow in the nitrogenase tree and do not help answer how these proteins evolved. The authors' reasoning for choosing these nodes is that they are likely compatible with the metal cluster assembly machinery of their chosen host organism, A. vinelandii. The …
Reviewer #1 (Public Review):
Understanding the evolution of nitrogenases is a very important problem in the field of evolutionary biogeochemistry. Ancestral sequence reconstruction at least in theory could offer insights into how this planet alerting activity evolved from ancestors that did not reduce nitrogen. But the very many components of the nitrogenase enzyme system make this a very challenging question to answer.
This paper now demonstrates the first empirical resurrection of functional ancestral nitrogenases both in vivo and in vitro. The nodes that are resurrected are very shallow in the nitrogenase tree and do not help answer how these proteins evolved. The authors' reasoning for choosing these nodes is that they are likely compatible with the metal cluster assembly machinery of their chosen host organism, A. vinelandii. The reader is left to wonder if deeper, more interesting nodes were tried but didn't yield any activity. As the paper stands, it proves that relatively shallow nitrogenase ancestors can be resurrected, but these nodes do not yet teach us anything very fundamental about how these enzymes evolved.
Technically, this work was no doubt challenging. Genome engineering in A vinelandii is very difficult and time-consuming. This organism was chosen because it is an obligate aerobe, which makes it easier to handle than the many anaerobic bacteria and archaea that harbor nitrogenases. It does make one wonder if this choice of organism is wise: the authors themselves note that it probably has a set of specialized proteins that allow the nitrogenase to be assembled and function in the presence of oxygen. This may limit A. vinelandii's potential future ancestral reconstructions deeper in the tree, which according to the authors' reasoning probably requires different assembly machinery.
The ancestral sequence reconstruction is done in two different ways: Two out of three reconstructions are carried out with what appears to be an incorrect algorithm implemented in older versions of RaxML. This algorithm is not a full marginal reconstruction, because it only considers the descendants of the node of interest for the reconstruction. The full algorithm (implemented e.g. in PAML and the newest versions of RaxML) considers all tips for a marginal reconstruction. The fact that this was called a marginal ancestral sequence reconstruction in RaxML's manual is unfortunate - as far as I understand it is in fact just the internal labelling of nodes produced by the pruning algorithm, which is not equivalent to a marginal reconstruction. In this specific case, it is unlikely that this has led to any fundamental issues with the reconstructions (as all are functional nitrogenases, which is to be expected in this part of the tree). For the shallower of the two nodes, the authors in fact verify that they get the same experimental results if they use PAML's full implementation of a marginal reconstruction (which yields a somewhat different sequence for this node). It would have been helpful to point this RaxML-related issue out in the methods, so as to prevent others from using this incorrect implementation of the ASR algorithm.
One other slightly confusing aspect of the paper is that it contains two different maximum likelihood trees, which were apparently inferred using the same dataset, model, and version of RaxML. It is unclear why they have different topologies. This probably indicates a lack of convergence. Again, this does not cast any doubt on the uncontroversial findings of this paper that shallow nodes within the nitrogenases are also nitrogenases.
-
Reviewer #2 (Public Review):
The authors convincingly show that their reconstructed ancestral nitrogenases are active both in vivo and in vitro, and show similar inhibitory effects as extant/wild-type enzymes.
The conclusion that, evolutionarily, there is a "single available mechanism for dinitrogen reduction" is not well explored in the paper. This suggests a limitation of using ancestral sequence reconstruction in this instance.
-
Reviewer #3 (Public Review):
In this work, the authors attempt to probe the constraints on the early evolution of nitrogen fixation, the development of which presented a key metabolic transition. Given that life on Earth evolved only once (to our knowledge) which aspects were necessary and which may have taken a different course are open questions. Are there alternative forms of life, metabolic networks, or even enzymatic mechanisms that could have replaced the ones we see today, or is the space of possible biologies limited? This manuscript tests the ability of ancestrally-reconstructed molybdenum-dependent nitrogenase complexes to support diazotrophic growth in Azotobacter vinelandii, as well as in vivo and in vitro activity, which all point towards a conserved mechanism for nitrogen reduction at least since proteobacteria divergence.
Reviewer #3 (Public Review):
In this work, the authors attempt to probe the constraints on the early evolution of nitrogen fixation, the development of which presented a key metabolic transition. Given that life on Earth evolved only once (to our knowledge) which aspects were necessary and which may have taken a different course are open questions. Are there alternative forms of life, metabolic networks, or even enzymatic mechanisms that could have replaced the ones we see today, or is the space of possible biologies limited? This manuscript tests the ability of ancestrally-reconstructed molybdenum-dependent nitrogenase complexes to support diazotrophic growth in Azotobacter vinelandii, as well as in vivo and in vitro activity, which all point towards a conserved mechanism for nitrogen reduction at least since proteobacteria divergence.
This is an ambitious project, requiring multiple techniques, systems, and approaches, and the successful combination of these is one of the major strengths of this work. Using parallel techniques is an important way to be certain that the overall results are robust, and an appropriate mix of in vivo and in vitro experiments is chosen here. The manuscript should serve as a useful model for how to combine phylogenetics and biochemistry.
The nature of ASR means that a solid phylogeny and/or understanding of how robust the results are to uncertainty in reconstructed states is essential since all results flow from there. The overall phylogenetic methods used are appropriate and the system is an apt one for the technique, but there is not quite enough detail in the methods to be certain of the results. Given that only the single maximum a posteriori sequence is assayed at every 3 nodes, this may have compounding results in that the sensitivity to uncertainty in the reconstruction is increased. The authors appropriately make qualitative rather than quantitative inferences, but some hesitation towards the overall results still exists.
The assumption that the Anc1A/B and Anc2 nodes correspond to ancestral states might be undermined by horizontal gene transmission, which has been reported for nif clusters. In particular, there may be different patterns of transmission for each element of the cluster. By performing reconstruction with a concatenated alignment, the phylogenetic signal is potentially maximized, but with the assumption that each gene has an identical history. Discordant transmission may cause an incorrect topology to be recovered.
Finally, I am unsure if ASR is the most appropriate approach to answer questions of contingency and alternative pathways for protein evolution. ASR may tell what nitrogenase millions or billions of years ago looked like, but it can only say what has already existed. If there are different mechanisms or metabolic pathways enabling nitrogen fixation that simply never came to pass, via contingency and entrenchment or simple chance, ASR would say nothing about them. It is true that a conserved mechanism would point towards a constrained space for evolving nitrogen fixation, but that does not directly address it.
Overall, despite these issues, the manuscript is compellingly written and the figures are attractive and clear, and help get the major narrative across. This work will be of interest to protein biochemists of evolutionary bent and microbial physiologists with an interest in the origins of life.
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