Substrate Specificity of an Aminopropyltransferase and the Biosynthesis Pathway of Polyamines in the Hyperthermophilic Crenarchaeon Pyrobaculum calidifontis

Round 1

Reviewer 1 Report

The work of Fukuda et al., is related to the production of polyamines by the hyperthermophilic archaea Pyrobaculum calidifontis and showed with soundness the production of thermospermine. Authors suggest, also, the production of norspermidine. Moreover, kinetic parameters of Pc-SpeE with different substrares are showed, and the crystal structure of the enzyme was solved. In this sense, lot of work was performed and it is not visualized in the title. I suggest a modification in the title, which give more information of the whole work.

The manuscript is well written, but some minor corrections must be performed:

• Different formats of running title, figures and tables. I would appreciate to check author guideleness.
• The number of methylene groups is mentioned in introduction (lines 52 and 53) and also in Figure 1. I suggest to mention and highlight, in introduction, that this information it is indicated in Figure 1.
• Line 55 a double space is between "from" and "decarboxylated".
• Line 82: authors mention a manganese catalase, but they do not indicate why it is important to include in the introduction. I suggest to remove.
• Different text format in lines 128 and 129.
• Lines 219-222: the information could be condensed in one sentence.
• Figure 3: Maybe the authors should include information related to what represent each clade in the phylogenetic tree. This could be more informative.
• Lines 351 and 385: in vitro must be in italics.
• There is no explicit conclusion.
• At the end of Funding section: it seems there is missing information.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript of Fukuda et al entitled „ Substrate specificity of an Aminopropyltransferase and the Biosynthesis Pathway of Polyamines in the Hyperthermophilic Crenarchaeon Pyrobaculum calidifontis” is a study on the polyamine biosynthesis pathway of P. calidifontis predicted based on enzymatic properties and crystal structures of an aminopropyltransferase from P. calidifontis (Pc-SpeE). After cloning the enzyme from genomic DNA and expressing in E. coli, four independent crystal structures with different ligand bindings were determined by using co-crystallization of protein solutions mixed with various combinations of substrates.

Although the manuscript is a well compiled report on the results, reviewer suggests adding further references to the Introduction on evolutionary diversification in polyamine biosynthesis [Minguet EG at al. Mol. Biol. Evol. 25(10) (2008) 2119–2128. (doi:10.1093/molbev/msn161)] and on the enigmas of biosyntheses of unusual polyamines in an extreme thermophile, Thermus thermophilus [Oshima T. Plant Physiol. Biochem. 48 (2010) 521–526. (doi:10.1016/j.plaphy.2010.03.011)]

Similarly, further reference is suggested to the Discussion on crystal structure of Pc-SpeE (and addition of a short statement to section 3.3). Although the thermospermine synthase from Medicago truncatula shares lower than 30% identity with Pc-SpeE, the crystal structure of Mt-TspeS (PDB: 6BQ2) indicated a homotetrameric (dimer of dimer) structure as well [Secula B et al. Biochem J. 475(4) (2018) 789–802. (doi:10.1042/BCJ20170900)].

Notes

• In section 2.3 the primers for cloning Pc-SpeE gene from genomic DNA are given but no further information is given on the origin of their sequences, please add this information to the manuscript somewhere.
• Line 134: in the name of IPTG “D-“ is a stereodescriptor which should be typed as small capital
• It would be desirable to show k(cat) values in Table 3 (instead or in addition to Vmax)

Since the paper contributes to understanding the polyamine biosynthesis pathway in archae based on convincing structural and biochemical analysis data, I warmly recommend the acceptance of this paper in Catalysis for publication. 

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The present paper brings the prediction of polyamine biosynthesis pathway of P. calidifontis on the basis of the enzymatic properties and crystal structures of an aminopropyltransferase from P. calidifontis (Pc-SpeE). As results, Pc-SpeE shared 75% amino acid identity with the thermospermine synthase from Pyrobaculum aerophilum, and recombinant Pc-SpeE could synthesize both thermospermine and spermine from spermidine and decarboxylated S-adenosyl methionine (dcSA).

In fact, the work is quite interesting and important for the area, since for this species, no work like this has been published yet. 
After a careful reading of the manuscript, I believe that the introduction falls short of the experimental work, and therefore, I believe that the authors should better explain the importance of studying the biosynthesis pathway of these polyamines and why the selectivity of the aminotransferase is so important.
In the discussion and results section, it should be clearer why this work is so important and innovative. There is not much discussion.
After the authors improve these points in the manuscript, I believe the work will be ready to be published.

Author Response

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Author Response File: Author Response.docx