Reviewer's report 1
Constantina Bakolitsa, Burnham Institute for Medical Research, CA 92121, USA
I find your revised version much improved. You have addressed my remarks adequately, with the exception of Figure 3 which I still think could benefit from a clearer representation.
I do not agree. This figure is complex because it describes the co-existence of two NAD(P)H – binding sites and their interactions with various residues involved in the stabilization of the coenzyme.
A couple of other points that might help further improve your manuscript.
1. You still need to check for spelling/grammatical typos.
Language errors have been corrected.
2. Your conclusion could benefit from having a few more sentences added summarizing your work prior to looking at future implications. Something perhaps like this: «The present study identifies a new coenzyme binding site in GDH4 with a potential regulatory role in GDH4 activity. The existence of such a site allows for the design of genetic engineering experiments that could potentially improve the efficiency of absorption and transportation of nitrogen. Other possible applications include enhancing the resistance of plants to environmental stresses such as dehydration, elevated CO2 levels and hypoxia. Further experiments will be required to address these issues.»
The conclusion has been modified in order to take into account this important remark.
Reviewer's report 2
Martin Jambon, The Burnham Institute for Medical Research, CA 92037, USA
Subject: This article presents a computational analysis of the glutamate dehydrogenase (GDH). This enzyme comes in 3 forms, classified according to its coenzyme specificity (NAD mostly: EC 22.214.171.124 or NAD-GDH, here denoted GDH2; NAD or NADP: EC 126.96.36.199, denoted GDH3; NADP mostly: EC 188.8.131.52 or NADP-GDH, denoted GDH4).
The analysis is concerned by the role of GDH4 in plants, as it plays a role in ammonium assimilation and its importance with respect to the glutamine synthetase/glutamate synthase pathway is unclear. To date, there is no crystal structure of GDH4, and the only known gene in plants comes from Chlorella sorokiniana, and leads to two isoforms.
Findings: The author conducted a sequence analysis of the GDH family and classified them into several groups according to their size and coenzyme specificity. A representative from the GDH3 subset was carefully chosen to serve as a template for building a theoretical 3D model of GDH4 from C. sorokiniana.
Besides analyzing functional motifs that are know from other GDHs, the author proposes and discusses the presence of a putative second NADPH binding site, based on the similarity with an aldehyde dehydrogenase.
Criticism: This study appears to have been conducted carefully, and brings an interesting perspective toward understanding the role and the regulation of the NADP-GDH in plants. This certainly should be published. This study does not generate new experimental results but proposes models that would be useful for future experimentations. This is why it would be interesting to see diagrams for proposed models that would explain structure-function relationships. In particular, is the role of the putative second NADPH binding site to activate the enzyme ? It would be interesting to draw rough scenarios of which cellular contexts could cause the activity or inactivity of the enzyme, and how it is possible that the enzyme is used more for ammonium assimilation than the opposite reaction.
The role of the putative second NADPH binding site is likely to activate the enzyme. The end of the discussion section has been re-written in that sense. It seems difficult, through a computational analysis, to draw such scenarios. I hope that this study can initiate further experimental approaches that will allow it. In particular, the first purification of GDH4 from plant, followed by its biochemical, enzymatic and cristallographic charaterization.
English language and typos.
Language errors have been corrected.
Reviewer's report 3
Sandor Pongor, International Centre for Genetic Engineering and Biotechnology, Italy
1. Generally speaking, computational analysis of protein families is always informative, in this respect the ms can be considered for publication, especially as part of a general review on the given protein family. I am not sure if a review supported with computational details is within the scope of Biology Direct. This work falls somewhat short of that aim, there is no systematic description of the pertinent literature, e.g.:
[a] Fontaine JX, Saladino F, Agrimonti C, Bedu M, Terce-Laforgue T, Tetu T, Hirel B, Restivo FM, Dubois F. Control of the synthesis and subcellular targeting of the two GDH genes products in leaves and stems of Nicotiana plumbaginifolia and Arabidopsis thaliana. Plant Cell Physiol. 2006 Mar;47(3):410–8. Epub 2006 Jan 17
[b] Masclaux-Daubresse C, Reisdorf-Cren M, Pageau K, Lelandais M, Grandjean O, Kronenberger J, Valadier MH, Feraud M, Jouglet T, Suzuki A. Glutamine synthetase-glutamate synthase pathway and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco. Plant Physiol. 2006 Feb;140(2):444–56. Epub 2006 Jan 11.
[c] Cruz C, Bio AF, Dominguez-Valdivia MD, Aparicio-Tejo PM, Lamsfus C, Martins-Loucao MA. How does glutamine synthetase activity determine plant tolerance to ammonium? Planta. 2006 Apr;223(5):1068–80. Epub 2005 Nov 16.
[d] Miflin BJ, Habash DZ. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops.J Exp Bot. 2002 Apr;53(370):979–87. Review.
[e] Stitt M, Muller C, Matt P, Gibon Y, Carillo P, Morcuende R, Scheible WR, Krapp A. Steps towards an integrated view of nitrogen metabolism. J Exp Bot. 2002 Apr;53(370):959–70. Review.
[f] Suzuki A, Knaff DB. Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism. Photosynth Res. 2005;83(2):191–217. Review
Four papers ([b], [c], [d] and [f]) suggested by Dr. Pongor were already added in the revised version (Ref. N° [32, 35, 6]and , respectively). Concerning the two other ones (not added), some papers, maybe older but more original, were prefered.
2. The work is about the structural features of GDH that can be predicted from computational analysis. It is not entirely clear to me what the main reason and the main goal of this analysis is. The author mentions, first in the title itself that a key role of GDH is suggested in this work. It is not apparent for me what this key role is, and how it can be related to the findings of this paper. One of the findings, the lack of the antenna region is not unique: non-mammalian GDH-s are generally knwon to lack the antenna regions.
First, as mentioned above by Dr Pangor himself, a computational analysis of protein families is always informative. Secondly, the goal of this study was to link some structural features of GDH4 to the reaction catalysed by this isoform (the assimilation of ammonium into glutamate). Third, the main finding is the putative second NAD(P)H – binding site together with four residues involved in the stabilization of the coenzyme. The increase of both the expression and the activity of GDH observed in some ammonium conditions is likely the result of interactions between the two sites, allowing allosteric regulation of the enzymatic activity of GDH4 in the absence of antenna.
3. The presentation of work is concise, the methodological details are put into the Appendix, which facilitates the reading of the work. The organization of the paper is not entirely clear. For instance, in the background there is a full summary of the conclusions. I do not see why this part belongs there. Some of the references are incomplete (publication year missing).
The text has been largely reduced and the message has been concentrated (most of the redundancy was removed). A global organization chart summarizing the key structural features of the central domain has been added in Figure 1. The alignment focalizes on the two coenzymes binding-sites. Four references have been added, mainly for argumentation of the controversy. I did not noticed any missing publication year in the revised version.
Reviewer's report 4
Frank Eisenhaber, Research Institute of Molecular Pathology (IMP), Vienna, Austria
The focus in this MS as described by the author is set on the role of GDHs in plants in the process of nitrogen assimilation. Targeting this goal with a sequence-analytic studies of various GDHs is problematic since it is known that these enzyme catalyze the reaction described on page 3 of this MS and the relative share of GDHs in the N-assimilation process is unlikely to be determined within the protein sequence of the GDHs themselves. Thus, this work will not contribute to this point. In this context, the reviewer wonders that the paper Glevarec et al. Planta (2004) 286–297 is not referred to.
I agree with the remark concerning the paper of Glevarec et al. When it was published, it seemed that GDH4 has no role in ammonium assimilation. Then, a lot of work has been published suggesting that it may have a key role. This paper is now mentioned.
The analysis of protein sequences of various subgroups of GDHs and the relationship of sequence patterns with function is another aspect of this MS; this question is more likely to be solved with the methods used in this work.
The collection of the sequence set that is the object of study is a critical point. It is the state of the art to collect the family by statistically rigorous similarity criteria applied on homologous sequence segments (in this case, apparently the central domain). For example, the BLAST/PSI-BLAST suite can be used:
Schaffer AA, Aravind L, Madden TL, Shavirin S, Spouge JL, Wolf YI, Koonin EV, Altschul SF. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 2001 Jul 15;29(14):2994–3005.
Altschul SF, Koonin EV. Iterated profile searches with PSI-BLAST – a tool for discovery in protein databases. Trends Biochem Sci. 1998 Nov;23(11):444–7.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–402.
It is unclear what kind of evidence supports the statements in the description lines, the similarity of the hydrophobic pattern and the conservation of critical functional residues are stronger arguments for structural and functional similarity. By ignoring non-annotated sequences, the author removes possibly important information about sequence variability and sequence knowledge about isoforms in some organisms.
As a next step, the family is subgrouped into clusters by sequence similarity criteria applied on the homologous segment. This is possible with programs such as CDhit, MCL or JACOP. Obvious cases can also be clustered manually. As distance criterion, the similarity determined with BLAST can be used.
Li W, Godzik A. cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006 May 26
Li W, Jaroszewski L, Godzik A. Sequence clustering strategies improve remote homology recognitions while reducing search times. Protein Eng. 2002 Aug;15(8):643–9
Sperisen P, Pagni M. JACOP: a simple and robust method for the automated classification of protein sequences with modular architecture. BMC Bioinformatics. 2005 Aug 31;6:216
Enright AJ, Van Dongen S, Ouzounis CA. An efficient algorithm for large-scale detection of protein families. Nucleic Acids Res. 2002 Apr 1;30(7):1575–84
The reviewer suggest that the subfamilies should resemble the GDH2-4 classification to some extent. Conservation of functional residues and, possibly, similarities in the sequence architectures within a subfamily (the sequence pieces outside the homologous domain) might support this clustering independently. Functional properties can possibly transferred within these subfamilies, e.g. the EC numbers.
I have carefully read Dr Eisenhaber's remarks, as well as its book chapter "Prediction of Protein function", and I agree with these accute observations. However, I feel that they do not apply to my work. Dr Eisenhaber describes a very elegant strategy for a completely unknown amino acids (or nucleotide) sequence for which one wants to discover its function trough its structure.
In this work, the dataset is clearly defined : all sequences correspond to the same enzyme (GDH) and moreover for most of them, the structure is known and even the enzymatic specificity at the EC number level. The goal of my work was to identify structural features specific of the isoform GDH EC 184.108.40.206 from plants in order to demonstrate its putative key role in ammonium assimilation.
Concerning the removing of non-annotated sequences, I do not agree. Keeping in the data set the sequences annotated as «putative», «unknown protein », etc..., would have almost decreased the precision of the information.
The conclusion about a second co-enzyme binding site is, at present, of speculative nature since a sequence pattern conservation detail and a 3D modeling study provide just plausibility.
It seems true for all computational analysis.