Peer review of: The chloroalkaloid (−)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate- (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The authors describe the discovery of a 2-oxoglutarate/Fe(II)-dependent halogenase of plant 
origin. The identified enzyme, named dechloroacutumine halogenase (DAH), catalyzes the terminal 
chlorination step in the biosynthesis of the chloroalkaloid (-)-acutumine in plants of the 
Menispermaceae family. The corresponding genes encoding DAH in Sinomenium acutum and 
Menispermum canadense were identified by comparative transcriptomic analyses versus the non-
producer Stephania japonica, by tissue specific analyses (root vs. leaf tissues) of all three plant 
species and by probing for iron(II)- and 2-oxoglutarate-dependent dioxygenases. 
Ordering synthetic genes for the identified enzymes, the authors were able to express functional 
DAH enzymes from S. acutum and M. canadense in E. coli and could show halogenation activity in 
vitro. Furthermore, the authors highlighted anion promiscuity of the enzymes SaDAH and gave 
evidence that alternative catalytic activities can be accessed by SaDAH and its variants (azidation 
and hydroxylation). 
The research is well described and clearly presented and the manuscript's findings are a valuable 
contribution to the field as the discovery of a freestanding 2-oxoglutarate/Fe(II)-dependent 
halogenases of plant origin are expanding the (so far very small) biocatalytic toolbox of these type 
of halogenases. 
However, prior to publication in Nature Communications, there are a number of concerns which 
need to be addressed: 

Major points 

(1) The authors show that the newly discovered enzymes exhibit a certain anion promiscuity such 
as for NaN3. To increase the depth of the study, it would be interesting to also learn more about 
the enzymes’ substrate promiscuity, for example with respect to related alkaloids such as 
hasubanonine. 

(2) The depicted LCMS analyses are extracted ion chromatograms. It would be important to also 
show a TIC of the in vitro chlorination to rule out other major side products. 

(3) Supplementary information / Supplementary Figure 11: Dechlorination of (-)-acutumine: 
Recorded spectrometric data should be shown, purity of obtained product should be described. 

(4) The authors should give a more detailed description in the main text how the proposed (-)-
acutumine biosynthetic pathway was elucidated (Figure S1). Did the differential expression 
analysis in fact only yield one possible enzyme sequence candidate for each biosynthetic step as 
suggested in Figure 1c and S2? 

(5) Fe(II)-dependent halogenases have been previously described to exhibit hydroxylation activity 
(as also observed by the authors for the DAH enzymes). Importantly however, regioselectivity of 
CH-activation may differ depending on the reaction chemistry (e.g. hydroxylation vs. 
halogenation) as selectivity is determined by the orientation of the substrate with respect to the 
HO-Fe-Cl (Mathews et al., PNAS 2009, 106, 17723-17728; Hayashi et al., ANIE 2019, 58, 18535-
18539). Whereas the authors do have an acutumine standard to which they compare the obtained 
MS and MS2 data, such a standard is not described for the azide and hydroxylated (side) products. 
Nevertheless, the authors postulate a structure for the obtained hydroxy and azido derivatives of 
acutumine (Figure 2a, Figure S9). Contrary to the author’s argument in the Figure legend S9, in 
light of the findings of above mentioned papers structural data is required to verify regio-and 
stereospecificity of the proposed products. 

(6) What is the total turnover number of the DAH enzymes? 

(7) Is there any evidence for the proposed gene duplication event? 

(8) The authors conducted transcriptome analysis of all three plant species but only in case of M. 
canadense, this analysis was performed on different plant organs (root, leaf, stem). Does the plant 
S. acutum also show a difference in acutumine production in roots versus leaf? 

(9) The authors should state more clearly, why SaDAH was chosen as the representative DAH for 
further analysis. 

Other (minor) points: 

(1) Page 2: “To identify the candidate enzyme responsible for the terminal chlorination, we probed 
several known classes of halogenases, including heme haloperoxidases16, vanadium-dependent 
haloperoxidases20, flavin-dependent halogenases18, S-adenosyl-L-Met-dependent halogenases21, 
methyl halide transferases16, and iron(II)- and 2-oxoglutarate-dependent halogenases 
(2ODHs)22.” 
This sentence is confusing. The authors should describe shortly in which way they “probed” the 
obtained transcripts for all of the enumerated enzyme classes. 

(2) Supplementary information: Protein expression and purification 
The authors should include a reference SDS-Page gel image to show purity of the wildtype DAH 
halogenases and the constructed variants. 

(3) Supplementary information: SaDAH-WT kinetic assay 
How was the internal standard curve (obtained by quenching of enzymatic reaction after 3h hours) 
verified? Did the authors compare with integrated peak areas of product solutions with known 
concentrations to ensure that conversion of substrate to product was indeed quantitative? 

(4) Supplementary Figure 2 depicts a differential expression analysis of M-canadense root versus 
stem transcriptomes. What is the difference / interpretation of this data compared to the data 
presented in the main article (root versus leaf)? 

(5) Page 3: The name of the species should be written out with first mentioning (Menispermum 
canadense) 

(6) The following sentence needs to be clarified, as in DAH enzymes alanine corresponds to the 
wildtype. 
“As predicted by this mechanism, mutating Ala226 back to Asp readily converts DAH to a 
hydroxylase” 

Reviewer #2 (Remarks to the Author): 

This is an excellent paper describing well executed research that led to the isolation and partial 
characterization of a truly novel 2-oxoglutarate- and iron-dependent dioxygenase that catalyzes 
the chlorination of a alkaloid substrate as the final catalytic step in the formation of the unique 
chloroalkaloid (-)-acutamine in plants of the Menispermaceae family. The strategy to identify gene 
candidates and the biochemical characterization of the isolated enzyme are clever and scientifically 
sound, respectively. The plant biology aspects of the work or generally limited to a comparative 
transcriptional analysis of the newly isolated halogenase, which displays a similar root- versus 
stem-enhanced expression compared with uncharacterized orthologs putatively encoding other 
alkaloid biosynthetic enzymes. Although most of these orthologs are likely to encode the proposed 
enzymes, this caveat should be better defined in the manuscript. It would clearly be nice to include 

a gene knockdown-type experiment to further confirm the physiological relevance of the enzyme, 
but this is clearly and generally difficult in non-model plant systems. 

From a biochemical perspective, it would be interesting to include empirical data showing the 
substrate specificity, or lack thereof, of the novel halogenase. Other basic aspects related to the 
characterization of the enzyme are strong, and the discussion is pertinent and informative. 

Reviewer #3 (Remarks to the Author): 

The authors reported the discovery of first enzyme of plant origin involved in halogenation, which 
is rare and would be an important biotechnological tool for expanding chemodiversity of plant 
natural products. I suppose that the impact and novelty of the finding reach the standard of the 
journal and are presented well generally. Only concern of mine is the amount of data, which seem 
fair for brief, but not for full, article. I hope that additional experiments suggested below would 
help to improve the study in that direction. 

1, They showed that SaDAH-G226H catalyzes the hydroxylation rather than halogenation. Is it 
plausible to confer the halogenation activity to related ODDs with a substitution HxG? This issue is 
of great interest, considering the generation of novel halogenation catalysts. 

2, In planta experimental evidences are lacking at all. Better to add the experiments technically 
possible to make the study more sound and lengthy enough, I believe. 

3, It would be better to deliver a reason (more enriched (-)-acutumine accumulation in the roots) 
before mentioning the selection of candidate transcripts based on their high expression in the roots. 

Reviewer #4 (Remarks to the Author): 

The work by Kim et al targets a rare yet important plant secondary metabolic pathway. 
Halogenated organic compound are frequent in pharmaceuticals and many other synthetic organic 
compounds, but are rarely found in plants. The authors targeted one these rare plant groups and 
used a simple yet elegant approach to identify candidate genes. They combined a simple 
comparative root versus leaf transcriptomics approach with candidate gene family selection and 
biochemical rational to identify a single candidate gene to catalyze the final chlorination reaction to 
produce the halogenated compound, called acutumine. Heterologous expression of the candidate 
protein in E. coli validated the predicted biochemical activity. Site directed mutatgenesis of an 
active site residue predicted to have changed the enzymes specificity from a hydroxylase to a 
halogenase also confirmed this prediction. Based on phylogenetic reconstructions the authors 
further show that the enzyme evolved from a plant enzyme (i.e. independently of its bacterial 
counterparts) rather than it was gained through lateral gene transfer. 

Overall these findings are significant for multiple reasons. First, having available a halogenase of 
plant origin opens new possibilities in plant synthetic biology and chemical engineering. Second, 
this is yet another example of convergent evolution in plant secondary metabolism and further 
cements the notion that few evolutionary changes may be sufficient to ‘create’ entirely new 
biochemical pathways. 

I have few suggestions that could improve the manuscript. The phylogeny is somewhat 
minimalistic, but serves the point that the enzyme evolved fairly recently through gene duplication 
rather than through lateral gene transfer. Expanding the species range within the Menispermaceae 
(and expanding it into other Ranunculales) would have more convincingly showed when the 

duplication happened within this family / order. 

The targeted mutagenesis (G226D) experiments clearly showed that G226 is necessary for 
halogenase activity, but it does not show that the D -> G mutation was sufficient to gain, at least 
some, halogenase activity. Introducing this mutation into the sister FLS-like sequence (or better 
into the immediate pre-duplication ancestor (which could have been reconstructed from the 
[expanded] phylogeny) would be an interesting experiment. 

Overall, the manuscript is written very well and I have essentially no editorial suggestions. Only 
the sentence “As predicted by this mechanism, mutating Ala226 back to Asp readily converts DAH 
to a hydroxylase” appears unclear. What does this refer to?