Biosynthetic study and production of novel fungal meroterpenoids
Takahiro MORI
Graduate School of Pharmaceutical Sciences, The University of Tokyo
ReportNon-heme Fe(II)/α-ketoglutarate (α-KG)-dependent dioxygenases are widely distributed in nature andcatalyze a variety of enzymatic transformations in both primary and secondary metabolism. In thisstudy, we performed biochemical and structural studies on the unique meroterpenoid dioxygenase
NvfE, which catalyzes the isomerization reaction without utilizing α-KG as a co-factor. Based on theseresults, we proposed a detailed mechanism for the NvfE-catalyzed orthoester formation reaction.

Research aims
Non-heme Fe(II)/α-ketoglutarate (α-KG)-dependent dioxygenases catalyze a wide range ofoxidative reactions including hydroxylation, desaturation, and ring formation1. Recent studies on thebiosynthesis of meroterpenoids from fungi have revealed that non-heme Fe(II)/α-KG-dependent
dioxygenases are one of the key enzymes involved in the structural diversification and complicationsof many meroterpenoids2. This class of enzymes utilizes α-KG and O2 as co-substrates to carry out aseries of oxidative transformations.
Novofumigatonin is a heavily oxygenated fungal meroterpenoid from Aspergillus novofumigatus
IBT 16806 (CBS117520), and possesses a unique orthoester group3. A biosynthetic study on
novofumigatonin revealed that two α-KG-dependent dioxygenases, NvfI and NvfE, are involved in theformation of the orthoester group. Functional analysis of these enzymes revealed that NvfI catalyzes
the endoperoxidation of asnovolin A to generate fumigatonoid A and NvfE catalyzes the endoperoxide
isomerization of fumigatonoid A to produce an orthoester group containing fumigatonoid C.
Interestingly, although NvfE shares sequence similarity with Fe(II)/α-KG-dependent dioxygenases, itcatalyzes an unusual isomerization reaction in an α-KG-independent manner. However, the detailed
mechanism behind this special enzyme is still unclear. A previous study suggested that NvfE might
belong to a new class of enzymes that gained isomerization activity by losing α-KG binding ability.
To better understand the mechanism underlying the NvfE enzyme activity and the late-stage
biosynthesis of novofumigatonin, we performed biochemical characterization and crystallization ofNvfE. Structure-based enzymatic analysis and mutagenesis studies were then conducted based on thecrystal structure and sequence comparison of NvfE with other characterized Fe (II)/α-KG dioxygenases.

Methods
NvfE was expressed in Escherichia coli, and the purified enzyme was used for enzyme reactionand crystallization. A cubic crystal was obtained in 400 mM potassium phosphate dibasic, 1600 mM
sodium phosphate monobasic, 100 mM sodium phosphate dibasic/citric acid pH4.2, and 8%
Jeffamine®-M600 at 20°C, with 10 mg/mL of enzyme. From this condition, the crystal structure ofapo-type NvfE with Fe(II) was successfully obtained at 1.85 Å resolution.

Results
First, to investigate the metal dependency of the enzyme, in vitro enzymatic reactions of wild-type
NvfE were performed with fumigatonoid A and various metal ions, including Fe2+, Mn2+, Cu2+, Co2+,and Ni2+. The results indicated that Fe is essential for NvfE activity and cannot be replaced by othermetal ions.
To understand the reaction mechanism, X-ray crystallography of NvfE was conducted. The overall
crystal structure of NvfE possesses a double-stranded β-helix fold and forms a funnel-like reaction
chamber conserved in a jelly roll barrel, as in the case of other Fe(II)/α-KG-dependent dioxygenases
such as AndA (PDB ID 5ZM3), which catalyzes two-step desaturation and isomerization in thebiosynthesis of anditomins4. NvfE exists as a homodimer and its dimer structure is constructed mainly
through hydrogen-bonding interactions between the residues from each monomer. Comparison of theactive site between NvfE and AndA revealed that the interaction of the conserved 2-His-1-Asp facial
triad with iron in the active site is nearly identical in both enzymes.
The main differences between the apo-type NvfE crystal structure and the AndA in complex withα-KG and the substrate lie in the three loop regions. The length of loop A, between the Ser70-Cys83
region, is shorter in NvfE compared to that of AndA. Loop A is highly flexible and encapsulates thesubstrates in the active site of AndA. From the crystal structure of AndA, the density of loop A can
only be clearly observed in the complex structure, which represents the closed conformation of AndA.
In contrast, the density of loop A can be clearly observed in the apo-type crystal structure of NvfE,even without substrate binding. Interaction between Y78 and E149 fixes the conformation of loop A
to form a closed conformation in the apo-type NvfE structure. Furthermore, loop B, between Pro288Gly294 from the adjacent monomer is known to participate in interaction with the substrate. Loop B
in NvfE is also shorter than that in AndA and other dioxygenases. In addition, there is a long loop Cfrom the adjacent monomer located close to the active site in the NvfE crystal structure. Thus, thesestructural differences, especially the closed conformation of NvfE without the substrate, could be thereasons for the unique function of NvfE.
To further understand why NvfE does not require α-KG as a cofactor, we performed mutagenesisexperiments. First, Y78A, Y78F, E149Q, and a double mutant, Y78F/E149Q, were constructed andanalyzed using in vitro assays. Unexpectedly, these mutants showed only slightly reduced activity incomparison with the wild-type enzyme. Further, no new product or oxidation product was found inthese mutants with or without α-KG. These results suggest that the interaction between Y78 and E149
as well as other interactions could be important for forming a closed conformation. Therefore, we also

constructed mutants of hydrophilic active site residues as D76A, D135A, S136A, S140A, S177A,
Y226A, and E261A. All mutants demonstrated activities comparable to that of the wild-type enzyme.
These results suggest that a single mutation is not significant for displaying the functional differenceof NvfE, and that these active site residues are not essential for catalytic activity. Therefore, thefunction of the enzyme should be fixing the endoperoxide moiety of the substrate close to the ironcenter for cleavage and rearrangement of the endoperoxide linkage.
Based on these observations, we propose that NvfE catalyzes the orthoester formation reaction,
which starts from the cleavage of endoperoxide that is mediated by iron. At the beginning, Fe(II) binds
to the C13 oxygen, which leads to the cleavage of the peroxide bond, generating an oxygen-centeredradical, which subsequently undergoes β-scission leading to the formation of a carbon-centered radical.
Finally, this intermediate is oxidized by Fe(III) to generate carbocation, which is quenched by tworounds of heterocyclization to form fumigatonoid C.

Conclusion
Our biochemical and structural studies of the unique meroterpenoid isomerase NvfE have unveiled
the detailed reaction mechanism of the orthoester formation reaction. To further understand thefunction of active site residues, we are now investigating double and triple mutants of NvfE as well as
the loop engineering of this enzyme. We are also working on the random mutagenesis of NvfE to alter
its substrate specificity, reactivity, and co-factor dependence. The engineered enzymes will beheterologously expressed in fungi harboring the novofumigatonin gene cluster enzymes, to construct a
platform for the production of novel unnatural compounds.

References
1.

White, M. D., and Flashman, E., (2016) Catalytic strategies of the non-heme irondependentoxygenases and their roles in plant biology. Curr. Opin. Chem. Biol., 31: 126-135.
2.

Matsuda, Y., et al., (2016) Unusual chemistries in fungal meroterpenoid biosynthesis. Curr. Opin.
Chem. Biol., 31: 1-7.

3.

Matsuda, Y., et al., (2018) Novofumigatonin biosynthesis involves a non-heme iron-dependent
endoperoxide isomerase for orthoester formation. Nat. Commun., 9: 2587.

4.

Nakashima, Y., et al., (2018) Structural and computational bases for dramatic skeletal rearrangement
in anditomin biosynthesis. J. Am. Chem. Soc., 140: 9743-9750.