Dear all,
just a side comment to Guillermo's and Debanu´s discussion, which deals
with the term "apo".
I got the text below from a reviewer, who—as an exception ;-)—was
absolutely right:
In enzymology, enzymes that require the aid of cofactors such as metal
ions or prosthetic groups (e.g. a heme group, etc.) are holo-enzymes. In
the absence of these groups, the enzyme is non-functional and is termed
apo-enzyme. A common mistake that can be detected lately in the
literature is to call a holo-enzyme that lacks a bound substrate or
product an apo-form. Please, replace apo form with unbound form
throughout the text except when dealing with forms lacking the catalytic
zinc ion.
Best regards,
Xavier
-------- Forwarded Message --------
Subject: Re: [ccp4bb] comparison of different protein states
Date: Tue, 31 Jan 2017 22:23:27 -0800
From: Debanu Das <debanu....@gmail.com>
Reply-To: debanu....@gmail.com
To: CCP4BB@JISCMAIL.AC.UK
Dear Guillermo,
I think the referee has a point because you are comparing two
different proteins to propose a model of 2 states even though the
enzymes are highly similar with conservation of key features. With 40%
id/60% similarity, even if core domain structure and length are
conserved, there are likely differences in loops, etc.? Are any such
structural variations in the vicinity of the functional site or in
regions that could impact approach or function of the active site? If
so, the apo and bound forms could also be due to intrinsic differences
between the two orthologs?
In the absence of additional supporting evidence, I suppose one way to
frame it would be to emphasize that you are proposing a model based on
available experimental evidence on closely related proteins and also
admit the limitations and unconfirmed/speculative nature of the
proposed model?
However, I think there may be something more interesting you can do
for supporting your theory in case there are other experimental
structures of closely related members of the same enzyme family. If
so, you could compare in detail additional apo forms of multiple
related enzymes and then point to the complex form as an outlier due
to its structural differences, which then may represent your proposed
model.
In addition, do you have assay/mutational analysis of your protein
compared to the other one? If so, and if they have similar activity
and full conservation of active sites, then that can also be used to
support your theory that the 2 enzymes are structurally and
functionally very similar and so the two structures are representative
of your proposed pathway/model.
I had a similar situation a few years ago in comparative structural
analysis of 2 nucleases. Active site residues were identical with the
same metal and overall architecture was similar despite other
structure/dimer differences. Referee insisted we mutate our active
site residues and compare, which we did.
Hope this helps.
Best,
Debanu
--
Debanu Das,
Accelero Biostructures
On Tue, Jan 31, 2017 at 9:40 PM, Guillermo Montoya
<guillermo.mont...@cpr.ku.dk> wrote:
Dear all,
first of all sorry for this off-topic question. I am requesting your help to
find some papers to convince one referee about the comparison
of two different protein states.
In our manuscript we show the crystal structure of an
enzyme. This structure represents the enzyme after catalysis in complex with
the product.
In the discussion we have superimposed the enzyme in the apo conformation
and the enzyme after catalysis in complex with the product
and we have commented the conformational changes observed
between these 2 states to propose a model.
The point of the referee is that this comparison is not valid because the
enzymes that we used in the comparison belong to different species.
They are not the same protein.
However, and this is stated by us in the figs and the manuscript, these two
proteins are
40% identical and 60% conserved, the polypeptide length is the same, and
the key amino acids and the domain structure are fully conserved. They are
obviously orthologs.
I´d really appreciate if you can send me some literature/information
to support our approach
Thanks a lot for your input
best
Guillermo Montoya, Prof., Dr.
Research Director, Protein Structure and Function Programme
Novo Nordisk Foundation Center for Protein Research
Faculty of Health and Medical Sciences, University of Copenhagen,
Blegdamsvej 3A, DK-2200 Copenhagen, Denmark
web: www.cpr.ku.dk
