Peer review of: Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrie (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript by Gao et al. examines the catalytic mechanism of cyanobacterial acyl-acyl carrier 
protein reductase, AAR, using AAR and ADO enzyme complexed structures. The protein structural 
analyses for the complexed crystals are to the best of my knowledge technically sound and 
performed correctly. However, several questions are raised in the interpretation of the data 
results. 

Major concerns: 
The authors suggest the catalytic process of AAR through the protein structural analyses and 
biochemical data. In this proposed mechanism, although active machinery has been explained 
through the analysis of protein structure and biochemical data, it do not appear to have clear 
information on when and how ADO separation and binding with AAR are performed. When the 
substrate is transferred from AAR to ADO, it may be necessary to elaborate the position change of 
the substrate, described as “rotameric switch” in this paper. 
Has the author tested the enzymatic activity of the AAR-ADO complexed crystals ? 
What is the gene ID for AAR and ADO? 

Correction: 
In fig S9, change NpADO to PmADO 

Reviewer #2 (Remarks to the Author): 

Biosynthesis of alkanes in cyanobacteria has attracted great attention as a substitute for 
petroleum-based fuels. Cyanobacteria produces alkanes using a two-step reaction involving two 
enzymes, AAR and ADO. Although the crystal structures of ADO have been already known, 
structure of AAR has been elusive. This paper for the first time reports the structure of AAR and 
those of the AAR-ADO complexes in which AARs bind various ligands. The results suggest the 
structures of the AAR-cofactor and AAR-substrate complexes and a possible channel from AAR to 
ADO for efficient delivery of an aldehyde. Finally, the authors proposed a catalytic cycle involving 
structural changes of AAR and binding/dissociation of ADO. Although the purpose of this work is 
important and interesting, there are many concerns as described below. 

Major concerns: 
1. One of the major concerns on this paper is the direction of the acyl chain in the acyl tunnel of 
AAR. The present crystal structures showed that the alkyl group, instead of the aldehyde group, of 
the acyl chain is pointed toward the inside of ADO. If so, however, the alkyl group enters deep 
inside the ADO protein, where the reactive center is located, and the aldehyde group of the acyl 
chain cannot reach the reactive center of ADO. This is unreasonable. Thus, the present results 
cannot clearly explain the mechanism of efficient delivery of an aldehyde product from AAR to 
ADO. 

2. Although AAR was previously suggested to have higher activity for acyl-CoA than for acyl-ACP 
(ref. 15), it was later demonstrated that AAR has a strong preference for acyl-ACP substrates over 
acyl-CoA substrates. Since the natural substrate of AAR is acyl-ACP, the authors should use acyl-
ACP rather than acyl-CoA for crystallization studies. Without the crystal structure of the complex 
between AAR and acyl-ACP, the mechanism of AAR catalysis cannot be solved. In this sense, the 
present report is preliminary and needs additional data. 

3. Unfortunately, the binding site of Mg2+ ion(s) was not determined in the present study, 
although this information is important in understanding the role of Mg2+ in AAR catalysis. 

4. The authors assumed that Mg2+ and K+ ions bind to AAR in the crystals. However, there have 
been no biochemical evidence showing that the metal ions do bind AAR. Therefore, it is required to 
confirm that Mg2+ and/or K+ really bind to AAR using ITC or other methods. Note that it is 
possible that K+ ions do not directly bind AAR but just enhance the solubility of acyl-CoA in the 
presence of Mg2+ ions. See Constantinides & Steim (1986) Arch Biochem Biophys. 250(1): 267-
270. 

5. It is also unfortunate that a long contiguous channel from AAR to ADO was not observed in the 
present crystal structures. 

6. To understand the activity of AAR and its mutants, it is necessary to measure kcat and Km 
values, instead of reporting just an octadecanal yield (see Table S2). Also, it is desirable to 
measure activities of all mutants used in the present study. 

7. To verify the AAR-ADO interface, the authors created three ADO mutants targeting the acidic 
residues of helix 7 and measured the binding affinity of AAR with them using ITC. The results 
showed that both the E200A/D201A double mutant and the E196A/E200A/D201A triple mutant of 
ADO completely lost the binding affinity to AAR. However, it is still unknown which of E200 and 
D201 is essential for the AAR binding. In addition, contributions of E196 and E208 are unknown. 
To clarify these issues, it is necessary to create four additional mutants, namely the E196A, E200, 
D201A and E208A single mutants and study the contribution of each acidic residue of ADOO on the 
AAR binding using ITC. It is also recommended to make some AAR mutants at positively charged 
residues, to confirm the importance of electrostatic interactions between AAR and ADO. 

8. The present results indicate that acyl-CoA and NADPH binds at the same site on AAR 
competitively. To verify this, it is required to confirm that acyl-CoA cannot bind to AAR 
simultaneously with NADPH using, for example, ITC. 

9. It is required to measure the binding affinity (Kd) of ADO with the Y247A and Y247F mutants of 
AAR, because Y247 of AAR forms a hydrogen bond with H7 of ADO. 

Other comments: 
10. In Fig. S9, W178 in SeADO is compared with R191 in PmADO. For this purpose, the authors 
used the PDB file of 4PGK for the structure of PmADO. However, this structure has the R191L 
mutation (not Val but Leu) and is not appropriate for the above purpose. The authors should use 
other PmADO structures that do not have mutation at R191 in Fig. S9 and Fig. 4. 

11. To solve the AAR(NADPH)-ADO structure, the authors soaked the AAR(thioester)-ADO crystal 
with NADPH. However, it is curious why the authors did not try to construct the AAR-ADO-NADPH 
ternary complex without addition of acyl-CoA. 

12. When presenting the ITC data, the authors only showed the Kd values (see Table S2 and Figs. 
S3 and S6). However, to check the validity of the ITC experiments, other parameters should also 
be shown, including delta_H, delta_S, and stoichiometry. 

13. The ITC data on the binding of AAR with stearoyl-CoA, shown in Fig. S6, indicate a 
stoichiometry of 1:0.5, but this value is not reasonable. 

14. In the ITC measurements, the authors prepared an AAR sample of 720 uM. However, since 
AAR is prone to aggregation (see ref. 15), there is a concern that AAR was aggregated, and the 
ITC measurements may not be properly carried out. If the authors have some tips to solubilize the 
AAR protein at high concentrations, please describe them in detail. 
15. The methods section should be described in more detail in Supplementary Information. To 
reproduce the purification of the AAR and ADO samples, it is required to describe, for example, 
components of media and antibiotics, timing of IPTG induction, cultivation time of E. coli, methods 
of determination of protein concentration and so on. In addition, please describe the amino acid 
sequence (22 residues) attached at the C-terminal region of AAR as a His-tag. 

16. The purity of the AAR and ADO proteins should be shown. For example, the data on SDS-PAGE 
after purification and the profiles of size exclusion chromatography using Superdex 200 should be 
attached in Supplementary Information. 

17. In the AAR(apo) structure, there were three flexible regions, called Fr-I, Fr-II and Fr-III. 
Although roles of Fr-I and Fr-III are discussed in the manuscript, nothing is discussed on Fr-II. 

18. Page 2, line 23-24: In the Abstract, the authors wrote that the two enzymes act in series and 
potentially form a complex that efficiently converts long chain fatty acids into hydrocarbons. 
However, this sentence is incorrect: instead of fatty acids, fatty acyl-ACP or fatty-acyl-CoA is 
converted into hydrocarbons. 

19. The authors should clearly describe which cyanobacterial species AAR and ADO are derived 
from. Line 78 describes that AAR for the apo form is from Synechococcus elongatus PCC 7942. Is 
the same AAR used for AAR-ADO complexes? Is ADO derived from the same cyanobacterial 
species? 

20. In Fig. S4c, the lines pointing to M21 are not properly shown. 

21. There are many typographical errors in the manuscript: 
- Line 78: Synechococcus elongates 7942 --> Synechococcus elongatus PCC 7942 
- Line 333: plasmid GKJE8 --> plasmid pGKJE8 
- In Fig. 2c, V8 is probably L8. 
- Line 2 of Fig. S1 caption: homologous --> homologues 
- Line 2 of Fig. S1 caption: elongatus 7942 --> elongatus PCC 7942 
- Line 3 of Fig. S1 caption: Nostocacea sp. PCC 7120 --> Anabaena sp. PCC 7120 
- The tile of Fig. S9: NpADO --> PmADO 
- Table S2: Kd value (uM) --> Kd value (microM)