Peer review of: Siroheme synthase orients substrates for dehydrogenase and chelatase activities in a (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript concerns investigations into the mechanism of CysG, the siroheme synthase 
multifunctional enzyme that transforms uroporphyrinogen III into siroheme. The enzyme performs 
methylations at C2 and C7 of the macrocycle before catalysing dehydrogenation and chelation 
reactions. The latter two reactions are performed in the N-terminal region of the protein within a 
single site. The authors have undertaken a significant amount of work including crystallography, 
mutagenesis and enzymology in order to try and compose a molecular overture of events to explain 
how this enzyme is able to perform the dehydrogenation and chelation reactions. Excitingly, the 
authors have managed to generate substrate and product complexes of the enzyme and from this are 
able to provide new insights into the action of the enzyme. This works makes a significant advance to 
the area and represents a fairly complete piece of work. The work complements on-going studies 
elsewhere that aim to understand how cellular metal availability is related to enzyme activity so this 
paper is rather timely. There are some comments the authors may wish to address below: 

1. Throughout text, replace typhimurium with enterica. Alternatively, for this serotype, Salmonella 
enterica subsp. enterica serotype Typhimurium, it can be abbreviated to Salmonella Typhimurium. 
Personally, I’d stick with S. enterica. 

2. In the abstract – it is stated “CysG’s single active site … “ but of course this is confusing. CysG has 
its methyltransferase active site, and is a dimer, so therefore technically there are 4 active sites?? 

3. I am not familiar with proponyl as a term – propionyl is the more traditional spelling. Also, vitamin 
does not require a capital V. On trivial points, the term porphine (P11) refers to a metal-free 
porphyrin, rather than an isobacteriochlorin. 

4. It is stated on Page 3 – that siroheme is more like vitamin B12 in terms of having propionyl and 
acetyl side chains. Given that B12 is ring contracted – a better comparison would be with F430. 

5. The results are well aid out and nicely explained. Given that the precorrin-2 density is so weak, I’m 
not sure that the authors can raw too much concerning its actual position and distortion – most of this 
is inferred. Similarly, I’m not sure they can, with real accuracy, say that the C15 position is located 
more closely located to the NAD than sirohydrochlorin, especially given that the NAD is modelled. 

6. In the discussion, I’d point out that the CysG/Met8p chelatases represent a unique class III of 
chelatase, structurally distinct from the class I and II chelatases (see Brindley et al, 2003, J Biol 
Chem. 278, 22388-95). 

7. I would try and compare the two aspartates (D104/262) found above the tetrapyrrole substrate in 
CysG to the residues found in CbiK/CbiX (two His; see Romão et al (2011), PNAS 108(1):97-102) and 
the ferrochelatases (His and Glu; Medlock et al 2007, PNAS 104(6):1789-93) 

8. Finally, to put this back into a cellular context – I’d try and relate what you have found with the 
need to be able to bind Fe within the cell and relate this to the available intracellular metal 
concentration – see Osman et al (2019), Nat Chem Biol 15: 241-249. 

Martin Warren 

Reviewer #2 (Remarks to the Author): 

The manuscript by Pennington et al. describes a very thorough study of the enzyme SiroHeme and its 
interactions with the changing prosthetic group. There are at least eleven diffraction and refinement 
data tables (Table 1 type reporting) with resolutions better that 2 Å and good refinement statistics. 

The amount and the quality of information is outstanding. Nevertheless, it is difficult to present this 
amount of information in a way that makes it easy for the reader. This reviewer thinks that reading 
pages of results on eleven structures (or more) is difficult. The manuscript would be highly improved if 
the format is changed to make the section after the Introduction a Results and Discussion. Combining 
these two sections early in the manuscript will allow the reader to understand the changes described 
in the present Results section as they are presented as opposed to learn about them and their 
implications only after all observed changes are described.. 

Reviewer #3 (Remarks to the Author): 

The manuscript presents X-ray crystal structures of Salmonella typhimurium siroheme synthase 
(CysG) liganded to substrates product and metals. CysG has a bifunctional active site that catalyzes 
the final two steps of siroheme production, NAD+-dependent dehydrogenation and iron chelation. 
Currently, it is not known how a single active site performs these different chemistries. Semi-
quantitative assay of complementation and of the two activities are presented to test the importance 
of active site residues using site-directed mutagenesis. The topic of the manuscript should be of 
interest to a broad readership on enzyme mechanisms, iron utilization, and metabolic pathways. 
Generally, the manuscript is well written and the assay work informative but a lack of information for 
the X-ray crystallographic methodology makes it impossible to properly assess the technical merit. As 
detailed below, the authors should present debiased omit maps and information about the B-factors of 
the ligands in order to confirm the presence of the ligands, which are the basis for the findings. 
Indeed, the electron density that is presented for the structure of the complex with precorrin-2 
appears very poor and raises serious concerns about this particular liganded structure. The authors 
might consider the following points (page numbers from PDF for review): 

Major concerns: 
1) The electron density shown in figure 2d,e,f is from 2Fo-Fc maps To adequately assess the ligand 
density a debiased polder or simulated annealing omit maps or composite omit maps should be shown 
here or in supplementary materials (and it would be best if they were in stereo view). Indeed, the 
density for the structure with precorrin-2 is extremely disconnected and although the authors discuss 
this point, without additional maps and information on temperature factors there cannot be confidence 
in the interpretation. The additional maps are of great importance - on the precorrin-2 global structure 
validation metrics from the PDB validation report are below average for the resolution in all but one 
category. Omit maps and the average B-factor of the precorrin-2 ligand compared to the average of 
the surrounding side chains should be presented. This should be done for all three liganded structures 
(B-factors of protein, solvent and ligands should be given separately in the Crystallographic Table). 

2) The type of dimer referred to as an X shape is more technically referred to as a domain-swapped 
dimer where one domain makes close contacts to a second domain from a different chain 

3) Page: 7- The authors state that “Sirohydrochlorin is the most tightly-bound, based on strong 
density and higher occupancy (Figure 2e).” It is not reasonable to make assessment of affinity based 
purely on occupancy and quality of density as the resolution of the X-ray crystal structures and 
competing molecules in the crystallization milieu have direct effect on both. 

4) Page 7- The authors state “Co-sirohydrochlorin is the most stable of the molecules but is not bound 
deeply in the open active site cavity so the density is even more diffuse than that for precorrin-2, but 
sufficient to allow us to assign the orientation of the central plane of the tetrapyrrole as nearly 
orthogonal to the precorrin-2 and sirohydrochlorin binding poses (Figure 2e).” I think Figure 3f is 

meant here and the density of Co-sirohydrochlorin shown in the figure looks stronger not more diffuse 
than that of precorrin-2. Again omit maps are essential here for the reader to assess occupancy and 
orientation. 

5) Page: 10- “Both R260A and R261A CysG are overall similar to wild-type, with an RMSD of less than 
1 Å after superimposition (Figure S2a).” As molecular replacement phasing is used then there should 
be some note that the rmsd value is biased somewhat by the use of the wild type structure in phasing 

6) Page: 11- The authors state “Indeed, superimposition of an NADH-bound subunit shows that C15 is 
close to the nicotinamide ring that will abstract the hydride to make sirohydrochlorin (Figure 4a). 
Flattening of the ring upon conjugation moves C15 away from the NADH (Figure 4b).” Can the authors 
please provide the distances here rather than saying “close to” and “away from”? It would also be 
important to have the coordinate error reported for each of these structures so that differences can be 
assessed in context. 

7) Page: 12- “In the open active site, the P133G variant has the same structure as the wild-type 
enzyme, suggesting that the interactions between the helical domain and the dimerization domain are 
important for preforming the closed active site. Can this be clarified- it is not clear how it follows from 
the previous statement that the in P113G the closed active site hydrophobic substrate binding 
platform is distorted. 

8) Page: 13- "when a loop including D104- A105-P106-K107 (DAPK loop) flips over to lock the 
tetrapyrrole in the active site (Figure 3b)." The text should call out Fig 5b. Also the legend to Figure 5b 
does not describe that the two loops from apo and bound structure are shown and does not indicate 
the color scheme for them. The wording of the corresponding paragraph should be clarified- I take it 
Pro 106 acts as a steric block to prevent methylated tetrapyroles at C20 from binding. But the wording 
outward makes it seem like the distance is increasing? Please clarify, 

9) The paragraph starting "One longstanding question about CysG’s function is how it binds to the 
metal ion for insertion and how the enzyme selects for iron over cobalt, given its ability to act as an 
iron and cobalt chelatase. D104 has been implicated in that activity" then goes on to describe the 
activity of D104 site directed mutants and their structures. Although the findings are consistent with 
the conformational changes described for the DAPK loop, it has nothing to do with the metal 
selectivity. The findings on these mutations are should be put in the appropriate context 

Minor typos etc. 
Throughout the manuscript proponyl group should be propionyl group (there is a carboxylate at the 
end). 

Page: 5 "of the numerous amino acids that makeup the active site" should read "of the numerous 
amino acids that make up the active site" 

Page: 6 
“substrate/product that persisted after backsoaking" should read for clarity "substrate/product and 
this color persisted after backsoaking” 

Page 7- “CysG is also a sirohydrochlorin cobaltochelatase, although selective for Fe2+”. This 
statement needs a reference 

Page 7- “Two more arginines derive from CysGA” please clarify as the previous sentence does not 
discuss Arg specifically, it is unclear why these two are discussed separately 

Page: 9- The authors point out “water-mediated hydrogen bonds to the amide hydrogens on P1141, 

A1281, G1301, and S132. The amide N on proline is not protonated in a peptide bond. 

Page: 14- Uro’gen III methylation is catalyzed by a dimeric SUMT in all known pathways that is 
homologous to SUMTs involved in Vitamin B12 biosynthesis" should probably read "Uro’gen III 
methylation is catalyzed in all known pathways by a dimeric SUMT homologous to SUMTs involved in 
Vitamin B12 biosynthesis” 

The legend for figure 4 does not describe the differential coloring of the structures overlayed. 

Figure S1a legend needs correction" Sirohydrochlorin would interaction with S128 in the wild-type 
enzyme" should read “ would interact with” 

In the crystallographic Table check sig figs- for instance for one structure resolution is given as 2.486 
- 2.4