Peer review of: A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript entitled “Rectifying the catalytic promiscuity: A conserved strategy of chalcone 
isomerase-like protein to correct chalcone synthase specificity” presents very interesting finding of 
the functional role of chalcone isomerase-like proteins (CHILs). It has been shown that CHI has 
evolved from a fatty acid-binding protein (FABP), but there is intermediate group (CHILs) between 
CHI and FABP in the phylogeny, and the function of CHILs (they are clearly neither CHI nor FABP_ 
are mystery for a long time. 
In this paper, the authors beautifully demonstrated that CHILs are bind to CHS to enhance THC 
production and decrease CTAL formation, and rectifying the promiscuous CHS catalysis. The 
authors presented a large bulk of experimental data to convincingly show that CHIL binds to CHS 
and act as rectifier to reduce the promiscuous CHS catalysis. Such function of proteins is rather 
unusual and would be of a great interest in the community of enzymology/biochemistry in general. 
I believe that the work presents highly important findings and suitable to publish in Nature 
Communications. 

I have only one problem in this paper. 
While I do not have any problem with all biochemical experimental data the authors presented. I 
have to say that the phylogenetic analysis they presented does not meet a standard to publish in a 
good journal. First, they have employed neighbor joining method to infer the phylogenetic tree, 
which is the most unreliable method among all other methods (it is simply quick and dirty method, 
and the common sense is that it is not a good method). I suggest rerun the phylogeny with more 
reliable and standard methods such as Maximum Likelihood or Maximum Parsimony (ideally few 
different methods), for example, IQ-tree (http://www.iqtree.org/) provides a nice interface for an 
non-expert to generate much more reliable tree for publication. I also cannot understand what the 
authors discuss what they found in the phylogenetic relationships from the tree. I suspect that 
they will see much different tree from what they present (so I would not comment much in the 
current text), but they should pub clear explanations about the phylogeny and what they discuss 
in the text (more clear figure caption). 

Reviewer #2 (Remarks to the Author): 

This manuscript reports exciting results. CHIL is known to stimulate flavonoid formation. Here the 
authors not only detect the physical interaction between CHS and CHIL in the flavonoid metabolon 
but also demonstrate that this interaction is PKS- and species-specific. Most importantly, CHIL 
greatly increases the formation of THC at the expense of CTAL. Rectifying the promiscuity of an 
enzyme via interaction with components of the metabolon is likely to apply to other pathways in 
plant specialised metabolism, which will stimulate research into this interesting field. The authors 
have used an array of techniques and have refined procedures employed. Before consideration for 
publication, the following aspects may be revised. CHIL sequences were amplified from a number 
of species. If the protein is encoded by a gene family, isoforms may differ in their interaction and 
rectifying potential. A 5- to 10-fold molar excess of CHIL over CHS was found to be highly efficient. 
Can the authors estimate the ratio present in the engineered E. coli strain, e.g. antibody-based? 
Co-expression may lead to an improved interaction compared to simple mixing and hence result in 
higher, possibly exclusive, THC production, provided the excess of CHIL is ensured. In this context, 
how to rule out that the observed 100 kDa band refers to two AtCHIL monomers on one AtCHS 
monomer? Previously, CHI was found to bind to CHIL Given the small size of CHIL, the ratio of the 
CHS products may be affected by additional binding of CHI, which could be tested by co-
expression of CHI in the engineered E. coli strain. In the co-precipitation experiment, 150 and 192 
proteins were able to specifically bind to the His6-target proteins. How specific is this binding, 
given the high numbers of proteins? After clarification of these aspects, a publication in Nature 

Communications can be taken into consideration. Minor points: line 64, please correct 
‘hydroxycinnamoyl’; line 65, please add ‘general’ phenylpropanoid.