Reviews of second PLoS ONE submission

Update: Here's our official response.

The review of the PLoS ONE paper we submitted March 15 came back April 5th and can be found below.  Very fast!  (Our first PLoS ONE submission, which we submitted February 23, is still under review.  The editor tells us he had a hard time finding people to review it).

In Denmark April 5, 6, and 9 are holidays so this is my first real chance to think about the reviews and here are my gut reactions (I'll post a link to our formal response here later).  

1. Yes, given the fact that this appears to be the first paper on high-throughput computational estimation of barrier heights for enzymatic reactions, the paper is by definition proof-of-principle.

We do state on page 13 that "The construction and optimization of mutant structures as well as the generation of energy profiles is automated so that the barrier height of one mutant can be estimated in ca. 24 hours on around 10 processors once the WT reaction path has been constructed."   However, given how central this is to the paper, we probably should expand on this a bit.

2. Well, as we mention, this is the subject of a paper we are currently writing and many of the experiments are not done yet.  However, its hard to see how the development and testing of a rather complex and novel computational strategy and the description and structural rationalization of activity data for dozens of mutants can be squeezed into the same paper. 

3. This is discussed at the end of page 8, where we note that this probably has to do lowest minimum of the enzyme-substrate complex not being found.  However, since the binding mode is very similar in all mutants this is likely a systematic error that does not affect the relative barriers significantly.  We should add a discussion of this in the manuscript.

4. General remark: true, but I don't see how to include all these effects in a high-throughput screening method, with the exception of using continuum solvation model (we should re-evaluate some of the computed barriers using COSMO single points along the reaction path).  However, binding affinity changes are generally not considered in current state-of-the-art computational studies of enzyme catalyses and good results are often obtained without considering protein dynamics.  The fact that we are interested in relative barriers should make these issues even less important.  But, true, no reason not to point this out in the manuscript.   We do state on page 2:

In order to make the method computationally feasible, relatively approximate treatments of the wave function, structural model, dynamics and reaction path are used. Given this and the automated setup of calculations, some inaccurate results will be unavoidable. However, the intend of the method is similar to experimental high through-put screens of enzyme activity where, for example, negative results may result from issues unrelated to the intrinsic activity of the enzyme such as imperfections in the activity assay, low expression yield, protein aggregation, etc. Just like its experimental counterpart our technique is intended to identify potentially interesting mutants for further study.

With regard to the structural model being arbitrary: we did test three different structural models (Figure 4) so I can't agree with that.

5. There is nothing in the method used to compute the barriers (i.e. no atomic specific force field parameters or things like) that is specific to this enzyme or even enzymes in general.

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PONE-D-12-07445
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, we feel that it has merit, but is 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 below:

1. The manuscript seems to be more a proof of principle
than a real validation of efficiency and performance.   Moreover, it is not completely clear
if the protocol is fully automatic or if it requires lots of manual intervention.


2. There is no experimental verification of the predictions.

3. The calculated barriers are significantly too low, suggesting that the methods used are not adequate for reliable predictions.

4. There is no consideration of effects of binding affinity changes, nor effects of protein dynamics, nor solvation. The choice of molecular model appears somewhat arbitrary.

5. It is not apparent that these methods will be generally applicable to other enzymes. More evidence and testing is required.

We encourage you to submit your revision within sixty days of the date of this decision.

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.

If you choose not to submit a revision, please notify us.

Yours sincerely,

xxx (name removed upon request)
Academic Editor
PLoS ONE

Reviewers' comments:

Reviewer #1: The manuscript by Hediger et al. presents a computational protocol for the efficient
calculation of the effect of enzyme mutations on the height of the barrier of the
enzyme-catalyzed reaction.  The approach is based on semiempirical methods and is
applied to amide hydrolysis as catalyzed by the lipase B of C. antarctica.
The topic is interesting.  The main conclusions seem to be supported by the results
presented (but see below).  The manuscript is clear.

Major remark:
The authors mention at the end of the Conclusions section that the
application of their protocol to "190 different mutants" will be published elsewhere.
As such, the present manuscript seems to be more a proof of principle
than a real validation of efficiency and performance.   Moreover, it is not completely clear
if the protocol is fully automatic or if it requires lots of manual intervention.

Minor remark:
pagination and publication year are missing in some references (e.g., 18 and 24).

Reviewer #2: This short paper describes tests of computational approaches for predicting barrier heights for amidase activity in a lipase. The work is preliminary. There is no experimental verification of the predictions. The calculated barriers are significantly too low, suggesting that the methods used are not adequate for reliable predictions. There is no consideration of effects of binding affinity changes, nor effects of protein dynamics, nor solvation. The choice of molecular model appears somewhat arbitrary. It is not apparent that these methods will be generally applicable to other enzymes. More evidence and testing is required.