Proteins and Wave Functions: 2nd reviews of A Computational Methodology to Screen Activities of Enzyme Variants

I've been really busy, so haven't kept up with the developments on this paper:

* The reviews arrived on September 24th and can be found below.

* I replied directly to the editor the same day:

Dear xxx
I have just received the reviews. While I believe we can formulate an appropriate response, given the fact that it took 4 months to get the last revision reviewed, we will only re-resubmit if you are willing to make a decision without soliciting further reviews and within a week of receiving the revised manuscript. Otherwise, we will retract the paper as we feel we can get it reviewed considerably quicker at another journal.
Best regards, Jan

The editor replied the same day:

Dear Jan:
I am willing to make a decision within a week of receiving your revised manuscript. I may decide to take such decision assisted by further review(s). This will not affect the time to take my decision.
Best regards
xxx

* We submitted the revised version (on arXiv) and this letter on October 5th

* The paper was accepted on October 15th (see below)

======

PONE-D-12-07445R1

A Computational Methodology to Screen Activities of Enzyme Variants

PLOS ONE

Dear Dr Jensen:

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, I feel that the manuscript is still not suitable for publication as it currently stands. Therefore, my decision is "Major Revision."

We invite you to submit a revised version of the manuscript that addresses the points of the reviewers. We encourage you to submit your revision within forty-five days of the date of this decision. We apologize for the delay in providing you with the reviews of the manuscript. When your files are ready, please submit your revision by logging on to http://pone.edmgr.com/ and following the Submissions Needing Revision link. Do not submit a revised manuscript as a new submission. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please also include a rebuttal letter that responds to each point brought up by the academic editor and reviewer(s). This letter should be uploaded as a Response to Reviewers file.

In addition, please provide a marked-up copy of the changes made from the previous article file as a Manuscript with Tracked Changes file. This can be done using 'track changes' in programs such as MS Word and/or highlighting any changes in the new document.

Yours sincerely,

xxx

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer #2: The authors have made some minor revisions to the paper to address some of the comments. This method will not be generally reliable for all enzymes: it will fail for mutations that cause significant structural changes, or that cause changes in mechanism (or change which step is rate limiting). It will fail for reactions for which the computational methods are unreliable (these methods are known to have significant failings for a wide variety of reactions, much more than just open shell systems and transition metals; conformation and structural properties are also problematic, e.g. even for simple peptides, and for hydrogen bonds). The proposed method will also fail for reactions in which changes in solvation are significant; it can only be successful where adiabatic mapping profiles can usefully be calculated and provide a useful approximation to the free energy barrier. Clearly also it cannot (and is not designed to) deal with changes in binding affinity (which may be significant, even at high substrate concentrations) or enzyme stability. The discussion should be expanded and clarified to make these points clear, so that the limitations of the proposed procedure are clear to anyone else who may wish to use the method. With those caveats, this type of proposed procedure may be useful to other workers. The reliability of the method for the lipase will only become clear when the authors complete and publish the comparison with experiment, but it will be useful for them to be able to refer to the full technical details of the procedure, so I recommend publication (with these minor revisions) so that their work is not delayed.

Reviewer #3: Hediger et al. propose a method to compute fast enzymatic barriers by optimizing, at a fixed distance length, the conformations obtained by geometric interpolation of reactant and product structure. Due to the large dimension of the system the authors decide to use PM6 semiempirical methods combined with MOZYME, instead of standard SCF.

As discussed by the authors a fast approach to screen the effect of mutations in the barrier will be useful for design. However the authors may show that, in spite of the approximations needed for a fast screening, their method is able to reproduce some experimental data.

A simple control should be the comparison of the obtained barrier with experimental ones. However the obtained value of 6.0 kcal/mol is much lower than the 15-20kcal/mol measured experimentally.

The authors claim that this is because they generate the initial enzyme-substrate (ES) complex based on the product (tetrahedral intermediate, TI) and therefore the active site does not fit optimally the substrate. A simple manner to confirm this is to compare the obtained barrier from the TI to the transition state (TS) with the values obtained for the trypsin (Ishida and Kato, 2003 JACS). Indeed, from the plot of Fig. 7, it seems that the reverse barrier is of aprox. 10 kcal/mol, that it is similar to the one of the trypsin.

It is expected that the reverse barrier is estimated better than the reaction barrier since, in this case, the TS is more similar to the product than to the reactant. However, if the active site of the enzyme is not optimal for the different species along the reaction path it can give serious problem in the barrier and reaction energies calculation, depending on the crystal structure used as an input. It can be explored if a fast optimization (even at classical level) of the binding site itself for the reactant, product and selected TS conformation can improve the barrier estimate.

The authors suggest that the effect of the low barrier is cancelled when computing the relative barrier of mutants against the wild type one, but this has to be proven. I do not find any explanation for the choice for the particular single mutants assessed. It should be better if the authors compare relative barriers obtained for few mutated variants with the experimentally determined kinetics parameters present in literature for that mutants.

Minor details.

A method in which a particular variable (in this case a distance between atoms) is used as a reaction coordinate implies that the user may have prior knowledge of the system. It could be also the case that there is some hidden variable (i.e. a dihedral) and this could lead to estimate wrongly the barrier. This aspect should be mentioned in the method applicability description (i.e. in page 14).

The tetrahedron intermediate acronym should be defined.

In page 2, line 49 there is a typo in 'efficiency'.

In page 3, line 53, in the sentence "the estimation of the reaction barrier of the reaction" one of the 'reaction' has to be deleted.

In page 4, line 31, 'CaLB' should be 'CalB' as in the rest of the manuscript.

In page 8, line 31, 'feasable' should be 'feasible'

===========

PONE-D-12-07445R2

A Computational Methodology to Screen Activities of Enzyme Variants

PLOS ONE

Dear Dr Jensen,

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE.

Your manuscript will now be passed on to our Production staff, who will check your files for correct formatting and completeness. After this review, they may return your manuscript to you so that you can make necessary alterations and upload a final version.

Before uploading, you should check the PDF of your manuscript very closely. THERE IS NO AUTHOR PROOFING. You should therefore consider the corrected files you upload now as equivalent to a production proof. The text you supply at this point will be faithfully represented in your published manuscript exactly as you supply it. This is your last opportunity to correct any errors that are present in your manuscript files.

If you or your institution will be preparing press materials for this manuscript, you must inform our press team in advance. Please contact them at ONEpress@plos.org.

If you have any questions, concerns, or problems, please contact us at plosone@plos.org, and thank you for submitting your work to our journal.

With kind regards,

xxx

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer #3: The authors have added some sentences to address the comments raised about the applicability of their method to other systems. More importantly, they refer in the text and make available an unpublished study in which they tested their method against experimental activities. From the application paper, the method appears promising to identify potentially interesting mutants, being able to discriminate, with exception of few variants, between 'very active' and 'very inactive' mutants. Therefore I recommend the publication.

One minor typo:

In page 14, line 15: 'reactivity apply: Identifying' should be 'reactivity apply: identifying'