Peer review of: ATP-dependent hydroxylation of an unactivated primary carbon with water (2023)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

The manuscript by Jacoby et al. describes the enzymatic activation of a primary carbon in the 
absence of O2. The major claims are novel and should be of interest to others in the community 
and the wider field. More specifically, the described series of reactions initiates the anaerobic 
catabolism of cholesterol in Sterolibacterium denitrificans. However, these reactions will surely 
represent a paradigm in the anaerobic activation of similar alkanes. For the most part, the 
experiments are clearly described, the data are clearly presented, and the data are convincing. 
Some additional characterization of the C26 hydroxylase would strengthen the manuscript. 

Specific comments: 

1. The manuscript needs to be carefully edited: it contains quite a few grammatical and stylistic 
errors. The following are cited as examples (the list is not exhaustive): “non-activated” should be 
“un-activated” (title, elsewhere); “no option” should be “not an option” (l. 49); “proceed” should 
be “proceeds” (l. 54); “During” should be “In” (l. 62); insert a comma at the end of line 83; “an 
artificial electron acceptor” (1.88); “analogues of it” should be “its analogues” (1. 97); “a so far 
unknown” should be “previously undescribed” (l. 104); “during” should be “using” (1. 125); 
“anaerobically conducted assays” should be “assays performed anaerobically” (1. 171); 
“putatively” should be “potentially” (l. 210); and “can hardly” should be “is unlikely to” (l. 223). 

2. The biologically relevant form of the reaction product is a carboxylate, not a carboxylic acid. It 
should be depicted and referred to as such throughout the manuscript (Figs. 1 and, l. 27, 1. 107, 
and elsewhere). 

3. The statement that “isoprenoid side chain is cleaved … prior to steran skeleton degradation” is 
incorrect (l. 63). Capyk et al. (J Biol Chem 286, 40717) provided evidence that these proceed 
concurrently to at least some extent. 

4. The first two sentences of the Results repeat the Introduction. The section should begin “To 
elucidate the enzymology responsible for production of CDO-26-oate” (l. 115). 

5. In Figure 4a, “DH5” should be relabeled as “S26DH1” (i.e., the identity of this enzyme needs to 
much clearer). 

6. The authors should determine the Mo, Fe, S, and heme content of their preparation of S26DH1. 
As it stands, the prosthetic groups of the enzyme are solely inferred from bioinformatics. 

7. The conditions used to achieve anaerobicity for the enzyme purification and characterization 
(e.g., kinetics) need to be described. This should include enough detail so that the reader is not 
obliged to look up additional papers. In addition, the authors should provide estimates of O2 levels 
in these manipulations. 



Reviewer #2 (Remarks to the Author): 

Boll and colleagues have discovered a sequential enzymatic process that enables hydroxylation of 
inert primary C-H bonds with water, which involves two molybdenum-dependent hydroxylases and 
one ATP-dependent dehydratase. This biological process is new and therefore this work represents 
interests and new insights for enzymologists and organic chemists. The authors performed 
thorough analytics and therefore the results and mechanisms in the manuscript are convincing. 
After evaluating the manuscript carefully, I think the manuscript needs some improvements at this 
stage before it can be accepted in top-tier Nature Communications. Concerns are shown below. I 
hope that the authors will find the comments helpful to improve the manuscript. 

1) In the introduction, the oxygen-independent enzymatic oxidation of C-H bonds is kind of 
limited. There are other enzymes that can perform similar reactions without the use of O2, e.g. 
peroxygenases, peroxidases, etc. It would be great if the authors can broaden the interest in the 
introduction. 
2) I suggest that in the supplementary NMR spectra the molecule structure should be included, as 
well as the assignment of the peaks, this is will be more straightforward to catch the information. 

3) The authors indicated that “the reaction cascade identified in this work allows in principle for 
any enzymatic water-dependent hydroxylation at a primary carbon next to a tertiary one.” Did the 
authors extend the substrate scope to see the specificity/genericity of the present enzymatic 
cascade? For example, what about the hydroxylation and oxidation of cholecalciferol and other 
similar compounds? 

4) The labelling of the compounds 1,2,3… are confusing, the compound number were sometimes 
not in bold, it would be great if the authors could label the compound more consistently and 
precisely like the way usually seen in organic synthesis. 

5) Is there any specific reason that the experiments were performed with crude extracts? In this 
way key information on the catalytic capability of the enzymes (e.g. TTN, TOF) are missing. 

6) The typo errors need to be checked: e.g. line 165, “my” should read “by”; in Figure 1 “26-OH-
CDO” was selected and the box was still there, etc. 



Reviewer #3 (Remarks to the Author): 

Specific Comments and Suggestions for Revision: 
The current study is the latest in a series of studies by M. Boll and co-workers aimed at elucidating 
the enzymatic reactions involved in steroid catabolism in the absence of oxygen by anaerobic 
bacteria. A new class of molybdenum-dependent steroid dehydrogenase enzymes capable of 
oxygen-independent hydroxylation of the C-25 group of cholesterol was first reported by Chiang et 
al., in 2008. In a recent paper by C. Jacoby et al. (mBio 9(3), 2018), four molybdenum-dependent 
dehydrogenase isozymes were overexpressed in bacteria, isolated and characterized. 

In my opinion, the study provides important new information regarding oxygen-independent 
hydroxylation reactions catalyzed by bacterial enzymes. A large amount of relevant data is 
presented in the supplementary section. This is a novel and interesting study. However I found 
some ambiguity in the characterization of the final two steps of the reaction sequence (as outlined 
below). The manuscript is well written. There are a few awkwardly worded phrases and un-
necessary sentences. Moreover, I found the use of semi-colons to be excessive. I think that the 
use of periods and shorter sentences would improve readability. 

Pg. 2 (Abstract), line 28, and elsewhere in the manuscript – I suggest deleting the word, 
‘unprecedented’, as this descriptor is un-necessary. 

Pg. 3 (Introduction), lines 39-47 ⸺ In my opinion, the Introduction section is too long. It seems 
to me that the first paragraph of the Introduction is un-necessary and could be deleted in order to 
simplify and shorten the introduction. 

Pg. 3 (Introduction), line 49 ⸺ I suggest replacing the words, ‘no option’, with, ‘not an option’. 

Pg. 3 (Introduction), line 54 ⸺ The word, ‘proceed’, should be written as, ‘proceeds’. 

Pg. 3 (Introduction), line 62 ⸺ The phrase, ‘… globally important bacterial degradation of 
cholesterol …’, should be justified. How and why is bacterial degradation of cholesterol important 
on a global scale? 

Pg. 4 (Introduction), line 65 ⸺ I suggest the phrase, ‘… during persistence in macrophages …’, in 
this sentence is confusing and un-necessary and should be deleted in order to simplify the 
sentence. 

Pg. 4 (Introduction), line 67 ⸺ I suggest adding replacing the abbreviation, ‘Cyp450s’, with, 
‘cytochrome P450 enzymes’. It should be noted that the widely used abbreviation for cytochrome 
P450 is CYP, not, cypP450. 
Pg. 4 (Fugure 1 legend), line 80 ⸺ It would be helpful if the abbreviation CDO, which is defined 
in the figure, was also defined in the figure legend. 

Pg. 5 (Introduction), lines 98 and 101 ⸺ The words, ‘rationale’ and ‘chaperone’ are mis-spelled 
here and elsewhere in the manuscript. 

Pg. 5 (Introduction), line 105 ⸺ I suggest simplifying the phrase, ‘… primary one would involve a 
so far unknown enzymology.’ by re-writing it as, ‘ … primary alcohol involves unknown 
enzymology’. 

Pg. 7 (Results), lines 111-121 ⸺ In my opinion, this paragraph does not belong in the Results 
section. It is perhaps more suitable for the Methods section. 

Pg. 9 (Results), lines 165-166 ⸺ It would be helpful if the units (eg. 1.2 ± 0.1 ATP) were 
specified. 

Pg. 9 (Results), line 170 ⸺ It would be helpful if the authors specified what was meant by ‘the 
latter’. What are they specifically referring to? 

Pg. 10 (Results), line 185 ⸺ I suggest re-writing the phrase, ‘… in cis to the C24 …’, as, ‘… in the 
cis position relative to the C24 …’. 

Pg. 10 (Results), line 188-190 ⸺ It seems to me that the sentence stating that the oxidation of 
the C-26 alcohol to a C-26 aldehyde needs some explanation because the authors state (on 
line172 and in Fig. 3) that cell-free extracts of Sterolibacterium denitrificans produced two 
products, 26-OH-DDO and DDO-26-al. It is unclear to me why the authors then state that 26-OH-
DDO was further oxidized to DDO-26-al. Are the authors suggesting that only the alcohol product 
was formed in the initial reaction and that the alcohol product is converted to the aldehyde product 
in a subsequent reaction? Is this oxidation reaction catalyzed by a new enzyme such as alcohol 
dehydrogenase or are both reactions catalyzed by S26DH1? To my mind, this sub-section is 
ambiguous 

Pg. 11 (Results), line 54 ⸺ I suggest inserting the word, ‘enzymes’, between ‘DH5-7’ and ‘may’.

Pg. 12 (Results), lines 214-215 ⸺ In my opinion, this sentence is worded awkwardly and should 
be re-written. 

Pg. 12 (Results), line 222 ⸺ It is unclear to me how ions obtained by TOF-MS identified the 
bands as the subunits of S26DH1. Do the authors mean that the masses of the ions correspond to 
the masses of the subunits? It would be helpful if the authors clearly explained the method of 
identification. 

Pg. 12 (Results), lines 228-235 ⸺ As noted above, the authors state (on line172 and in Fig. 3) 
that a cell-free extract produced two products, 26-OH-DDO and DDO-26-al, yet the authors now 
imply that S26DH1 first produced 26-OH-DDO and then catalyzed its dehydrogenation to DDO-26-
al. The dehydrogenation of an alcohol to an aldehyde is typically catalyzed by an alcohol 
dehydrogenase. Are the authors now suggesting that S26DH1 catalyzes separately a hydroxylation 
reaction and an alcohol dehydrogenase reaction? Is there evidence that S25DH1-4, which are 
related enzymes, can catalyze both a hydroxylation reaction and an alcohol dehydrogenase 
reaction? I think a more complete explanation is needed. 

Pg. 13 (Results), lines 236-258 ⸺ I found this section of the Results to be confusing. If I 
understand the section correctly, the authors are suggesting the involvement of two enzymes, an 
alcohol dehydrogenase and an aldehyde dehydrogenase. The alcohol dehydrogenase catalyzes the 
dehydrogenation of the C-26 alcohol to an aldehyde, which seems to contradict the statement on 
the previous page regarding S26DH1, while the aldehyde dehydrogenase catalyzes the 
dehydrogenation of the C-26 aldehyde to a carboxylic acid. Have the authors isolated and 
identified this new aldehyde dehydrogenase from E. coli? Again, I think a fuller explanation is 
needed. 

Pg. 15 (Discussion), line 287 ⸺ It is not clear to me what, ‘it’, (i.e. If it would proceed …) refers 
to. It would be helpful if the authors specified what was meant by ‘it’. What are they specifically 
referring to?