Structure function and engineering of multifunctional non-heme iron dependent oxygenases in fungal meroterpenoid biosynthesis

Non-heme iron and α-ketoglutarate (αKG) oxygenases catalyze remarkably diverse reactions using a single ferrous ion cofactor. A major challenge in studying this versatile family of enzymes is to understand their structure–function relationship. AusE from Aspergillus nidulans and PrhA from Penicillium brasilianum are two highly homologous Fe(II)/αKG oxygenases in fungal meroterpenoid biosynthetic pathways that use preaustinoid A1 as a common substrate to catalyze divergent rearrangement reactions to form the spiro-lactone in austinol and cycloheptadiene moiety in paraherquonin, respectively. Herein, we report the comparative structural study of AusE and PrhA, which led to the identification of three key active site residues that control their reactivity. Structure-guided mutagenesis of these residues results in successful interconversion of AusE and PrhA functions as well as generation of the PrhA double and triple mutants with expanded catalytic repertoire. Manipulation of the multifunctional Fe(II)/αKG oxygenases thus provides an excellent platform for the future development of biocatalysts.

1. The introduction suggests that these NHI enzymes are mostly involved in fungal meroterpenoid biosynthesis -actually they are very widely distributed and NHI rearrangements are relatively common.
2. I found the lack of detailed chemical mechanisms (e.g. traditional arrow pushing!) in the actual MS disappointing. The textual descriptions of the mechanisms are good, but support from clear diagrams for both the WT and mutant reactions (e.g as shown partially in Fig S1) would improve the impact and readability of the MS for non-expert readers. In particular discussion of stereoselectivity (i.e the faces over which the iron-oxo moiety has access) would be pertinent.
Reviewer #2 (Remarks to the Author): The authors compare AusE and PrhA, two non-heme oxygenases that catalyze distict reactions in terpenoid biosynthesis. The authors successfully crystallized the two enzymes and performed mutagenesis that swapped activities of the two enzymes, and also generated enzyme mutants with new activity. The manuscript is well-written and easy to follow. All required information for protein structure is present. This work is an important demonstration of how these iron containing enzymes can be manipulated and is suitable for Nature Comms. I have the following minor comments: page 5: "Despite numerous attempts, AusE could not be crystallized in the apo-form or in complex with Fe(II)." Did the authors try anaerobic conditions for this? I am not suggesting that the authors attempt this in a revision, but it would be informative to know if all crystallization attempts were performed in air. page 14: reference 22 is to a software program, which I think is a mistake Figure 3: Can the authors add structures to this figure, so that it is not required to go to one of the other figures to match the compound number to the structure? I think that it is essential to include a phylogenetic tree of fungal non-heme iron oxygenases that shows the placement of AusE and PrhA in a larger context. I would expect that these two enzymes would be closely related.
Reviewer #3 (Remarks to the Author): Overall I thought this was a well written and interesting manuscript describing biochemical, structural and engineering studies on an unusual oxygenase structured in terpenoid biosynthesis. The manuscript is generally well written (needs a final proofing). Although, there is a previous structure for one of the oxygenases, the current work is much more substantial and provides new insights. Overall, I'm in favour of publication with a few minor modifications, mostly to the figures.
Some of the Chemdraw type figures could be larger e.g. p28.

Supp fig 1, or an edited version thereof could be in main text.
Label N-/E termini on figures 3,4,5 Important to add in solution data on oligomerization status (I may have missed this), i.e. using gel-filtration or SEC MALs. Discuss role of the C-terminus in the text.

Specific points:
P3 para 2 -'highly identical' to 'very similar' Perhaps mention structure function studies on bacterial α-KG oxygenases in the introduction, e.g. work has been done on the bifunctional enzymes of cephalosporin biosynthesis (though I note mentioned in Discussion)

P5 -To investigate….
What is the oligomerization state of the protein in solution -gel filtration/SEC-MACS data should be provided. P6 -what exactly is meant by suprafacial? (and free standing).
Please label chair/boat as appropriate in Supp Fig 9   P7 -'direct contact' -give distance P8 -Define the assays being used (and in legend to Table 1) (it is important these analyse product formation as described in methods).
P10 -I don't think its correct to imply the initial H-abstraction controls the reaction -it's not always rate limiting and the products can vary even with the same initial abstraction.

Response to Reviewers' comments:
REVIEWERS' COMMENTS: Reviewer #1 (Remarks to the Author): This MS reports new and very detailed information both about the mechanisms and about the rational engineering of non-heme iron enzymes active in secondary metabolism. This class of enzymes is not as well understood as, for example, cytochrome P450 oxygenases, but are widespread and involved in fascinating and useful oxidative transformations including complex rearrangements in plants, bacteria fungi and marine organisms. The work has been performed to a very high standard and the MS is presented also at a very high level. The work concerns two parallel NHI enzymes involved in the oxidative processing of fungal meroterpenoids. The expression, purification and crystal structure determination is very difficult and the high quality results in this area are rarely achieved, and thus the methods and results reported will be of interest to many involved in structural determination as well as biosynthetic investigations. The mutants generated shed significant light on the mechanisms of the oxidations and how the enzyme controls the balance between all the different possible modes of oxygenation and rearrangement. The experiments are clearly designed and the results are generally clearly shown in the MS and supporting data. The MS tells a strong and coherent story and I found no significant scientific problems in the data interpretation or conclusions. Some minor points could be addressed.
1. The introduction suggests that these NHI enzymes are mostly involved in fungal meroterpenoid biosynthesis -actually they are very widely distributed and NHI rearrangements are relatively common.
Response: Thank you for the instructive comment. According to the suggestion, we have changed the sentence to "Non-heme iron and α-ketoglutarate (αKG; also called 2-oxoglutarate) dependent dioxygenases are widely distributed in nature, and play a major role in diversifying the molecular scaffold." (page 3, line 6 from the top).
2. I found the lack of detailed chemical mechanisms (e.g. traditional arrow pushing!) in the actual MS disappointing. The textual descriptions of the mechanisms are good, but support from clear diagrams for both the WT and mutant reactions (e.g as shown partially in Fig S1) would improve the impact and readability of the MS for non-expert readers. In particular discussion of stereoselectivity (i.e the faces over which the iron-oxo moiety has access) would be pertinent.
Response: According to the suggestion, we substantially revised Supplementary Figure 1. Fig S1 a, b, c, and d

Reviewer #2 (Remarks to the Author):
The authors compare AusE and PrhA, two non-heme oxygenases that catalyze distict reactions in terpenoid biosynthesis. The authors successfully crystallized the two enzymes and performed mutagenesis that swapped activities of the two enzymes, and also generated enzyme mutants with new activity. The manuscript is well-written and easy to follow. All required information for protein structure is present. This work is an important demonstration of how these iron containing enzymes can be manipulated and is suitable for Nature Comms. I have the following minor comments: page 5: "Despite numerous attempts, AusE could not be crystallized in the apo-form or in complex with Fe(II)." Did the authors try anaerobic conditions for this? I am not suggesting that the authors attempt this in a revision, but it would be informative to know if all crystallization attempts were performed in air.

Response:
We are sorry for the confusion, but in the original text, we had already stated that "All of the following procedures were performed under anaerobic conditions." (page 19, line 3 from the top).
page 14: reference 22 is to a software program, which I think is a mistake Response: Thank you. We have changed into the appropriate reference "21" according to the suggestion (page 14, line 24 from the top). I think that it is essential to include a phylogenetic tree of fungal non-heme iron oxygenases that shows the placement of AusE and PrhA in a larger context. I would expect that these two enzymes would be closely related.
Response: According to the suggestion, we have newly added Supplementary Figure 16 of a phylogenetic tree.

Reviewer #3 (Remarks to the Author):
Overall I thought this was a well written and interesting manuscript describing biochemical, structural and engineering studies on an unusual oxygenase structured in terpenoid biosynthesis. The manuscript