Peer review of: Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript “Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase” by 
C.-L. Chen et al. reports the structural studies on mevalonate decarboxylase from a vancomycin 
resistant enterococcus, Enterococcus faecalis (MDDEF). The authors succeeded in solving not only 
the apo-structure but also the structures with the substrate mevalonate diphosphate (MVAPP), 
MVAPP and an ATP analog AMPPCP, MVAPP and ADP, or MVAPP and ADP/BeF3-. As observed with 
the structures of other MDDs, the MDDEF structures took open or closed conformations, which 
allowed the authors to suppose the reaction mechanisms of the enzyme through substrate binding 
and phosphoryl-transfer, which is the first half of the MDD reaction. It is quite impressive that the 
authors could verify the binding positions of magnesium ions in the catalytic center of the enzyme. 
To my knowledge, this is the first discovery for this enzyme family and also the information 
needed to reveal detailed catalytic mechanism of MDDEF and to develop inhibitor molecules 
specific to the enzyme. Because human also possesses MDD, only such specific inhibitors can be 
utilized as antibiotics for VREs. The metal binding sites are confirmed with anomalous dispersion 
observed in the analysis of the crystals soaked with MVAPP and ADP in the presence of cobalt ion, 
instead of magnesium ion. This study is basically an extension of their previous studies on the 
structures and biochemical properties of MDDEF, but it clearly made a substantial progress. The 
manuscript is readable, and all the data look sound and persuasive. The reviewer, however, still 
has several concerns with the manuscript. First, the authors proposed the substrate binding 
mechanism based on the open-close change observed in the enzyme structures through the 
bindings of different substrates or their analogs. The proposed mechanism fits well the structural 
changes observed with MDDEF, but not with other MDD structures previously reported. For 
example, the alfa10-beta4-loop was in the closed position in the MDDEF structure with 
MVAPP/ADP/BeF3-/Mg2+, but in the open position with MVAPP/AMPPCP/Mg2+, thus the authors 
concluded that the former structure represents the pre-phosphoryl transfer state, while the latter 
does an earlier state. In contrast, the loop, and also the Lys residue corresponding to Lys187, 
were reported to be at the same positions in MDD from an archaeon Sulfolobus solfataricus 
(MDDSS) not only in similar substrate-binding structures but also in its apo form (pdb: 5gmd, 
5gme, 4z7c). The discrepancy casts question if the induced-fit mechanism proposed in this paper 
is general for MDDs or not. Additional discussion based on the comparison with the previously 
reported MDD structures is needed. Secondly, the mechanism of the ATP-dependent 
decarboxylation of MVAPP described in the manuscript, in which Asp282 acts as a base that 
deprotonate 3’-OH of MVAPP, has been controverted by recent finding of a MDD-homolog kinase 
that lacks the corresponding Asp residue but can catalyze 3’-phosphorylation of mevalonate 
(Vinokur J.M. et al., 2015, Protein Sci.), and also by a mutagenic study on MDDSS, which led the 
proposal of a hypothesis that Asp residue is required in the decarboxylation step (Motoyama K. et 
al., 2017, J. Biol. Chem.). If the authors want to refute the new hypothesis, they should perform 
QM/MM calculation study based on the structure of MDDEF binding MVAPP/ADP/BeF3-/Mg2+ or 
MVAPP/AMPPCP/Mg2+, which will clarify the protonation states of the substrates and catalytic 
residues and might support the less-persuasive discussion in page 15-16. 

Minor concerns: 
1) Page 2, line 7: large conformational “change”? 
2) Page 3, the 1st paragraph: The MEP pathway is found primarily in bacteria, not in prokaryotes. 
Archaea, another prokaryotes lineage, have the MVA pathway. 
3) Page 3, line 8: privarily > primarily 
4) Page 17, lines 19-: The method utilized for site-directed mutagenesis is obviously based on the 
QuickChange method. Appropriate references such as Zheng L. et al. (2004) Nucleic Acid Res. are 
needed. 
5) Page 19, line 10: Instead of 100 nM MDDEF, a higher amount of the mutant enzyme was 
considered to be used. Please show the amount. 
6) Page 21, lines 19-20: ligands “were” manually placed 

7) Supplementary Table 4: The table seems not showing the angles of alfa-helices, but their 3D 
positions. I am afraid that the title and legend are confused with those in Supplementary Table 3. 

Reviewer #2 (Remarks to the Author): 

In the manuscript entitled "Visualizing the Enzyme Mechanism of Mevalonate Diphosphate 
Decarboxylase" by Chen et al. (Manuscript NCOMMS-19-27996-T) the authors describe that two 
Mg2+ ions are bound to the enzyme preloaded with substrate and ATP. By this sequential binding, 
the active site is shaped for catalysis. 
The authors used X-ray crystallography to visualize the Mg2+ ions plus structures with transition 
state analogues. The resolution of the diffraction data is very high and the quality of the refined 
structures is very good. Surprised by the localization of the two Mg2+ ions, the authors generated 
structure-based variants of the MDD protein. 
To the reviewer’s point of view the current version of the manuscript is very well written and 
comprehensive. Nevertheless, I have the impression that most of the techniques described were 
already very well established in the lab and for instance described in earlier publications (Chen et 
al. (2017) JBC. For instance, the crystallization system has been described in the JBC publication. 
Probably in many instances, the described methods could be drastically shortened. Most of the 
designed variants have already been shown, to have functional implications. It is not proven, that 
the proposed mechanism is valid in other organisms than S. faecalis as well. In particular, the 
beta10-alpha4 loops show very little conservation. What is the function of R192? It seems to be 
conserved in bacteria, where as in higher organisms it seems to be a strict threonine residue. Is 
the catalytic side in both monomers identical? 

Minor points: 
- Throughout the manuscript, there is a mixture of one-letter and three-letter code for amino 
acids. 
- Page 5, line 13: Space is missing 1.6 M 
- Page 7, line 7: “extra density”. Please be more precise “extra electron density” 
- Page 7, line 19: probably “…next section” is meant 
- Page 19, line 8: Space is missing after 30 
- Page 19, line 18: please remove dot after Mus 
- Figure 1, panel a: I do not think that the topology diagram is necessary. The overall structure of 
MDDs is very well known 
- Figure 6, panel a: please carefully check the numbering of the alignment. I see some 
inconsistencies between the numbers of amino acids mentioned in the body of the text the 
provided numbering in the figure. Names of organism should be in italic.