Article

Cite This: J. Am. Chem. Soc. 2018, 140, 9743−9750 pubs.acs.org/JACS

Structural and Computational Bases for Dramatic Skeletal Rearrangement in Anditomin Biosynthesis Yu Nakashima,†,‡ Takaaki Mitsuhashi,†,‡ Yudai Matsuda,†,§,‡ Miki Senda,|| Hajime Sato,⊥,# Mami Yamazaki,⊥ Masanobu Uchiyama,*,†,# Toshiya Senda,*,||,□ and Ikuro Abe*,†,¶

† Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Department of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, China || Structural Biology Research Center, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan ⊥ Graduate School of Pharmaceutical Science, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-8675, Japan # Cluster of Pioneering Research (CPR), Advanced Elements Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan □ Department of Materials Structure Science, School of High Energy Accelerator Science, The Graduate University for Advanced Studies (Soken-dai), 1−1 Oho, Tsukuba, Ibaraki 305−0801, Japan ¶ Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

*S Supporting Information

ABSTRACT: AndA, an Fe(II)/α-ketoglutarate (αKG)-dependent enzyme, is the key enzyme that constructs the unique and congested bridged-ring system of anditomin (1), by catalyzing consecutive dehydrogenation and isomerization reactions. Although we previously characterized AndA to some extent, the means by which the enzyme facilitates this drastic structural reconstruction have remained elusive. In this study, we have solved three X-ray crystal structures of AndA, in its apo form and in the complexes with Fe(II), αKG, and two substrates. The crystal structures and mutational experiments identified several key amino acid residues important for the catalysis andprovidedinsightintohowAndA controls the reaction. Furthermore, computational calculations validated the proposed reaction mechanism for the bridged-ring formation and also revealed the requirement of a series of conformational changes during the transformation.

■ INTRODUCTION mechanisms for many αKG-dependent enzymes remain poorly As an excellent designer of small molecules, nature has evolved understood, as even the mechanism for the dehydrogenation, although it is apparently simple, is still controversial.6 See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. enzymes that perform diverse chemical transformations, often Therefore, it is important to utilize several different approaches

Downloaded via HIGH ENERGY ACCELERATOR RESRCH ORG on October 3, 2018 at 03:54:22 (UTC). with the aid of a variety of cofactors and cosubstrates, thus to clarify the reactions catalyzed by αKG-dependent enzymes, generating the complex and intriguing frameworks of natural ff products.1 Non-heme iron and α-ketoglutarate (αKG)- which will facilitate future bioengineering e orts with this class dependent enzymes are a widespread and major class of of enzymes. enzymes involved in both primary and secondary metabolism.2 AndA, involved in the biosynthesis of the fungal α They are mostly engaged in simple oxidative reactions, such as meroterpenoid anditomin (1), is an KG-dependent enzyme hydroxylation and dehydrogenation, but some atypical that catalyzes two consecutive reactions to construct a unique 4c fi reactions are catalyzed by this class of enzymes, including bridged-ring system (Figure 1). AndA rst accepts − the endoperoxidation by FtmOx1 (FtmF) and the epoxidation preandiloid B (2) as a substrate to introduce the C C double by AsqJ.3 Moreover, some αKG-dependent enzymes exhibit bond between C1 and C2 to yield the enone preandiloid C isomerase or epimerase activity, although they can also work as (3), which is further utilized as the substrate for the second oxidative enzymes.4 For the catalysis by Fe(II)/αKG-depend- reaction, in which it undergoes an intriguing skeletal ent enzymes, there is a strong consensus that the oxidative rearrangement to produce andiconin (4). Although the first decarboxylation of αKG first generates a highly active ferryl- oxo species, which then abstracts a hydrogen atom from a Received: June 9, 2018 substrate to initiate the reaction.5 Yet, the detailed reaction Published: July 4, 2018

© 2018 American Chemical Society 9743 DOI: 10.1021/jacs.8b06084 J. Am. Chem. Soc. 2018, 140, 9743−9750 Journal of the American Chemical Society Article

meroterpenoid pathways, such as PrhA (PDB ID: 5YBM, RMSD of 0.91 Å for the 202 Cα atoms).8 Next, the binding modes of the metal cofactor, the cosubstrate αKG, and the substrate 2 or 3 were investigated by soaking them in the AndA apo crystal under an anaerobic atmosphere. As a result, we obtained two more crystal structures of AndA, complexed with Fe/αKG/2 (PDB ID: 5ZM3) and Fe/αKG/3 (PDB ID: 5ZM4) at 2.25 and 1.95 Å resolutions, respectively (Figures 2B−E, S1, and S2 and Table S2). In the active sites of both structures, unlike the apo structure (Figure S3), the iron is present at the 2-His-1-Asp facial triad site (His135, Asp137, and His213) conserved among the αKG-dependent dioxygenase family members,9 while the αKG is coordinated to the iron in a bidentate Figure 1. Structure of anditomin (1) and AndA-catalyzed two-step manner, together with the facial triad and a water molecule reactions. The genuine “reducing agent” required for the second fi (w1), to form an octahedral iron complex (Figure 2D and E). reaction to provide andiconin (4) has yet to be identi ed for the in The αKG also interacts with the His79, Gln132, Thr173, and vivo reaction. Arg224 residues through hydrogen-bonding networks (Figure 2C). The quaternary complexes including substrates (2 or 3) reaction is a common dehydrogenation event, the conversion showed that both substrates are bound to AndA in an almost from 3 to 4 is an atypical isomerization. Our previous study identical fashion (Figures S4 and S5, RMSD of 0.19 Å for the revealed that the in vitro enzyme reaction of AndA requires 263 Cα atoms) and that they are located on the opposite side ascorbate as an essential factor, which probably reflects the fact of the facial triad, across from the ferrous ion (Figure 2D and that AndA functions as an isomerase.4c However, the E). As often seen in this class of enzymes, the substrate-binding mechanisms for the synthesis of the bridged ring and how pocket is constructed by the engagement of both monomers of the enzyme allows for this drastic structural rearrangement AndA: the loop between α2 and β3 (loop 58−76, loop A) have yet to be elucidated. from one monomer and the loop between α7′ and α8′ (loop In this study, to obtain the structural basis for the AndA- 261′−281′, loop B′; hereafter the primes designate residues catalyzed reactions, we solved the X-ray crystal structures of from the other chain of that without primes) from the other AndA complexed with iron, αKG, and the individual substrates are adjacently located to form a binding site, together with (2 and 3), which revealed the binding modes of the substrates Arg239 on another loop (Figures 2B, S6, and S7). Arg239 also and suggested the requirement of the ferryl-oxo species interacts with the carboxyl group of Glu66. Interestingly, loop isomerization prior to the hydrogen abstraction. Further A was observed only upon substrate binding (Figures 2A,B and mutational studies revealed some key residues specifically S6), indicating that it is highly flexible and that it serves as a lid important for the second reaction to produce 4. We also that encapsulates the substrates in the active site of AndA. This performed computational calculations of the rearrangement to lid-like region appears to recognize the D/E rings of the construct the bridged ring and successfully provided a rationale substrates, as suggested by the hydrogen bond networks for this conversion. observed in this area: two carbonyl oxygens (O3 and O5) both interact with the phenolic hydroxyl group of Tyr272′ as well as ■ RESULTS AND DISCUSSION the backbones of Gln67 and Ile70, via one water molecule (w3 Crystallographic Studies on AndA. To obtain insight or w4), while O4 is hydrogen bonded with two water into how AndA participates in the oxidative rearrangement molecules (w2 and w3), which in turn interact with the reaction, we sought to crystallize AndA for X-ray diffraction carboxyl group of Glu66 and the backbones of Lys63 and experiments. Since our initial attempt to crystallize the full- Gln67 (Figures 2D,E and S7). Meanwhile, the terpenoid length AndA was not successful, we prepared a truncated moiety (the A/B/C rings) of the substrates is relatively loosely protein lacking eight residues at the N-terminus (Met1-Tyr8), bound to the enzyme, since the interaction between O1 and which is predicted to be a disordered region. The truncated Asn121 is the only hydrogen bond detected in this region AndA was treated with EDTA prior to crystallization to (Figure 2D and E). completely remove any residual iron, and the metal-free In line with the common reaction mechanism for αKG- enzyme was then crystallized under anaerobic conditions, dependent dioxygenases, the αKG in the active site should leading to the X-ray crystal structure of AndA in its apo form at initially undergo oxidative decarboxylation to generate the 2.5 Å resolution (PDB ID: 5ZM2; Figure 2A and Table S2). highly active ferryl-oxo species, in which the oxo group is The crystal structure revealed that AndA exists as a symmetric located trans to His135. It was previously thought that this homodimer, constructed via several hydrogen bond networks ferryl-oxo species directly abstracts a hydrogen atom from a at the interface of the two monomeric units (Figure 2A). This substrate, as proposed for the well-studied αKG-dependent observation is consistent with the apparent molecular weight of enzyme TauD.10 However, recent spectroscopic and computa- AndA in its native state predicted by a gel filtration experiment. tional studies indicated that the isomerization (or rotation) of AndA possesses a double-stranded β-helix fold and forms a the oxo group, which proceeds with a low energy barrier, is funnel-like reaction chamber conserved in the jelly roll barrel, required before the hydrogen abstraction in the catalysis by as commonly seen in αKG-dependent dioxygenases (Figure several αKG-dependent enzymes.11 Thus, we sought to 2A).7 Accordingly, AndA exhibits a highly similar overall investigate the possibility that the isomerization event occurs structure to other αKG-dependent enzymes involved in fungal in the AndA-catalyzed reactions. Although it is impossible to

9744 DOI: 10.1021/jacs.8b06084 J. Am. Chem. Soc. 2018, 140, 9743−9750 Journal of the American Chemical Society Article

Figure 2. (A, B) Overall structures of (A) AndA-apo and (B) the AndA-Fe/αKG/3 complex. (C−E) Close-up view of (C) the αKG-binding site in the AndA-Fe/αKG/3 complex and the substrate-binding sites in the (D) AndA-Fe/αKG/2 and (E) AndA-Fe/αKG/3 complexes. The αKG molecule and the substrates 2 and 3 were modeled into composite omit maps (mFo − DFc map) contoured to 3.5σ. Important lengths are shown as dashed lines with distances labeled (average values for the four subunits in the asymmetric unit, units; Å). precisely determine the position of the oxo group, it would be AndA/Fe/αKG/2 complex, the distances from C1 or C2 to w1 close to the water molecule (w1) coordinated trans to His135 are 4.4 and 4.0 Å, respectively, while those to the carboxylic and to the carboxylic oxygen of αKG bound trans to His213 oxygen of αKG are 3.3 and 3.7 Å, respectively (Figure 2D), before and after the rotation, respectively. Therefore, we suggesting that the isomerization could occur to facilitate the measured the distances between these oxygen atoms and the hydrogen abstraction. For the second reaction, a hydrogen carbon atoms of the substrates from which a hydrogen atom atom at C12 is initially abstracted,5c and the oxo rotation could potentially be abstracted. In the first reaction to yield the seems to be more important than that in the first reaction, enone 3, a hydrogen atom at C1 or C2 of 2 should be since the carboxylic oxygen of αKG is located much closer to abstracted at the beginning of the reaction. In the quaternary C12 than w1 (3.5 vs 4.9 Å) in the quaternary AndA/Fe/αKG/

9745 DOI: 10.1021/jacs.8b06084 J. Am. Chem. Soc. 2018, 140, 9743−9750 Journal of the American Chemical Society Article

Figure 3. Computed reaction pathways and potential energy profiles of route A and route B. Three-dimensional representations of 7a, 7b, 7c, and 7d are shown. The C8−C2′ length in 7d is represented by dashed lines with the distance labeled (units, Å).

3 complex (Figure 2E). Collectively, our crystallographic study abstraction from C12, we identified two possible multistep suggests the requirement of the oxo group isomerization, but reaction pathways (routes A and B) for the dramatic skeletal this should be confirmed by future studies. rearrangement from 5 to 8, including (i) C8−O2 bond On the basis of the above discussion, the α-oriented cleavage, (ii) C12−C5′ bond formation, and (iii) C8−C2′ hydrogen at C1 would initially be abstracted in the bond formation, accompanied by several conformational dehydrogenation reaction to afford 3. To complete the changes (Figures 3 and S10 and Tables S4 and S5). The desaturation, the vicinal hydrogen at C2 could be abstracted, energy diagram suggested that both routes are thermodynami- followed by a diradical recombination to introduce the C−C cally and kinetically favorable: (i) the activation barriers are all double bond (Figure S8A). However, a recent mechanistic low enough for the reactions to proceed smoothly at ambient study of the AsqJ-catalyzed desaturation indicated alternative temperature, (ii) the entire energy profile descends as the mechanisms for this type of reaction: after the generation of reactions proceed, and (iii) the overall exergonicity is very the substrate radical, it could then undergo (i) the oxygen large (ΔG = −19.8 kcal/mol). Notably, the geometrical rebound and following dehydration or (ii) the oxidization by features of the computed 5 are in good agreement with those Fe(III) to a cationic species and subsequent deprotonation, to of the experimentally obtained 3 cocrystallized with AndA. We install the double bond.6b At this moment, none of the three found that the intermediate 7a and the subsequent cascade possibilities could be ruled out, but given the structural reactions to afford 8 had identical geometry and energy in both similarity between AndA and AsqJ, AndA could also use the routes. However, the key intermediate 7a can be reached from same strategy for the desaturation as that of AsqJ (Figure S8B). the reactant 5 via two different pathways. In route A, the C8− Computational Calculations of the AndA-Catalyzed O2 bond cleavage occurs prior to the C12−C5′ bond Rearrangement. We then performed a density functional formation, via the transition state (TS) TS_5−6 to give the theory (DFT) calculation (UB3LYP/6-31+G(d,p)) to obtain exomethylene 6, with a large stabilization energy (ΔG = −19.4 deeper insight into the reaction pathway and the mechanism kcal/mol), due to an intrinsically stable tertiary carbon radical. for the rearrangement to provide 4. First, we confirmed that The activation energy for the C−O bond cleavage is only 6.6 the 3D structure and conformation of the substrate 3 used for kcal/mol. Conversely, route B starts with 5-endo-trig (anti- the calculation are essentially the same as those of 3 in the Baldwin) cyclization to produce the α-oxy carbon radical cocrystal (Table S3 and Figure S9). After the initial hydrogen intermediate 6′, with an activation barrier of 23.4 kcal/mol.

9746 DOI: 10.1021/jacs.8b06084 J. Am. Chem. Soc. 2018, 140, 9743−9750 Journal of the American Chemical Society Article

The more than 15 kcal/mol difference in the energies of the first steps between routes A and B translates into an over 1015- fold difference in the reaction rates at room temperature; hence, route A should predominate over route B. From the intermediate 6 (route A), an intramolecular macrocyclization can occur smoothly along the intrinsic reaction coordinate with an activation barrier of 13.0 kcal/mol, to give the key intermediate 7a. After several conformational changes (7a → 7b → 7c → 7d) with very small activation barriers, the C8 carbon radical of the B ring in 7d approaches the potent Michael acceptor carbon center C2′ to complete the skeletal rearrangement, generating the most stable radical intermediate (8) with a large stabilization energy (ΔG = −12.3 kcal/mol). In the process of the conformational changes, the radical intermediates (7a, 7b, and 7c) can be interconverted with relatively low barriers. This is consistent with the X-ray crystal structure, as the A/B rings are relatively loosely bound to the enzyme in terms of the hydrogen bonding between the substrates and the enzyme (Figure 2D and E). To further increase the reliability of the above-proposed mechanism, we also investigated the effects of the hydrogen bonds on the radical rearrangement reaction by means of the theozyme (theoretical enzyme) calculation,12 in which some key interactions between the substrate and the enzyme are considered for the calculation. The rearrangement reaction proceeds on the B/C/D rings, and therefore, the hydrogen bonds with the A/E rings are too far from the reaction point to affect the rearrangement reaction. On the other hand, the hydrogen bonding of O-3/O-5 on the D ring with Ile70 and Tyr 272 via a molecule of water could electronically affect the radical rearrangement reaction. Thus, we carried out the DFT calculation with a molecule of water around O-3 and O-5 (Scheme S1), and it turned out that these hydrogen bonds do not greatly change the geometries of the intermediates/ transition states or the activation energies of this rearrange- ment reaction (Tables S6 and S7 and Figure S11). Thus, it is most likely that the bridged-ring synthesis proceeds through the route A (Figure 3 and Scheme S1). Mutational Experiment and Biochemical Character- Figure 4. LC-MS profiles of the products from in vitro enzymatic ization of AndA. To investigate how AndA is engaged in the reactionsoftheAndAmutantswith(A)2 or (B) 3.The rearrangement and to examine the importance of the amino chromatograms were extracted at m/z 413.233 (for 2; blue trace) acid residues constituting the active site, we performed and 411.217 (for 3 and 4; red trace). WT = wild type AndA. NC = mutational experiments on selected residues with side chains negative control. involved in the substrate binding, namely, Glu66, Asn121, Arg239, and Tyr272′. First, the point mutation of Asn121 requires a significant structural rearrangement. Since both (N121A) resulted in reduced activity, but did not completely Arg239 and Tyr272′ interact with the lid-like region, the abolish the conversions (Figure 4A), suggesting that the abolishment of the second reaction after the introduction of hydrogen bonding between Asn121 and O1 is not crucial for mutations to these residues could be attributed to the the reactions to proceed. Among the residues in the lid-like destruction of the lid function and the exposure of the D/E region, the mutations of Glu66 (E66A, E66D, and E66Q) only rings, which undergo significant structural rearrangements, to yielded insoluble proteins, which hampered the evaluation of the external environment. Collectively, AndA could play a the importance of this residue in the catalysis. Nevertheless, crucial role to protect the highly reactive radical intermediates the mutageneses of Arg239 and Tyr272′ provided intriguing by housing them in the closed active site, thus allowing the outcomes. The Y272F mutant exhibited activity comparable to intriguing skeletal rearrangement to proceed without quench- that of the wild-type enzyme, but all of the other constructed ing during the transformation. mutants, R239A, R239M, R239 V, and Y272A, produced only Finally, we performed biochemical characterization of AndA the dehydrogenated product 3 and lost the ability to synthesize and its mutants. Consistent with our previous in vitro 4 when 2 was used as a substrate (Figure 4A). Consistently, 3 enzymatic reaction with the wild-type AndA,4c the reactions was not further transformed into 4 when the above-mentioned by the mutants that produce 4 required ascorbate as an four mutants were used for the reaction (Figure 4B). In essential factor (Figure S12), which could be attributed to the contrast to the first desaturation reaction, which proceeds prediction that ascorbate is utilized to finalize the reaction. relatively inside the substrate-binding pocket, the second Interestingly, ascorbate is absolutely essential even for the reaction occurs near the entrance of the pocket and also mutants that are only able to perform the first-round reaction

9747 DOI: 10.1021/jacs.8b06084 J. Am. Chem. Soc. 2018, 140, 9743−9750 Journal of the American Chemical Society Article to produce 3 (Figure S12), although the dehydrogenation ■ MATERIALS AND METHODS reaction does not apparently require a reducing agent. This General Procedures. Solvent and chemicals were purchased from observation that ascorbate is indispensable for all the chemistry Hampton Research (CA, USA) and Wako Pure Chemical Industries, by AndA indicates that ascorbate has more function(s) than Ltd. (Tokyo, Japan), unless noted otherwise. Preandiloid B (2) and just reducing the product radical. One possible explanation preandiloid C (3) were prepared according to the previous work.4c would be that the productions of 3 and 4 are significantly Oligonucleotide primers were purchased from Eurofins Genetics uncoupled from the αKG oxidation and that ascorbate is (Tokyo, Japan). Sequence analyses were performed by Eurofins inevitably required to maintain the oxidation state of iron, but Genetics. fi this needs to be further clarified in future studies. We also Enzyme Expression and Puri cation for Crystallization. The N-terminal truncated andA gene was PCR-amplified from the measured the kinetic parameters for AndA and its mutants 4c (Table 1). The K values for both 2 and 3 increased after the previously prepared full-length andA-pET28a, with the primer m pairs andA-9-293-f and andA-9-293-r (Table S1). The DNA fragments were digested with NdeI and BamHI and ligated into Table 1. Steady-State Kinetic Parameters for AndA and Its pET28a using T4 DNA ligase (Wako Pure Chemical Industries, Ltd.). Mutants Constructed plasmids were introduced into Escherichia coli RosettaII-

−1 (DE3)pLysS (Novagen), and the transformants were cultured with kcat/KM (s − μ protein substrate k (s−1) K (μM) μM−1) Luria Bertani medium supplemented with 12.5 g/mL chloramphe- cat M nicol and 25 μg/mL kanamycin at 37 °CtoanOD of 0.6. The ± ± 600 wild type 2 6.31 0.14 52.6 9.7 2.00 cultures were further incubated at 16 °C for 18 h after 0.3 mM ± ± N121A 5.00 0.10 84.0 10.7 1.00 isopropyl β-D-1-thiogalactopyranoside addition. The cells were R239A 2.13 ± 0.11 70.1 ± 28.0 0.48 pelleted by a 20 min centrifugation at 6000g and lysed by sonication R239V 3.09 ± 0.12 75.8 ± 22.3 0.68 in buffer A (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5% (v/v) R239M 3.05 ± 0.07 85.5 ± 14.0 0.59 glycerol, and 5 mM imidazole). After the centrifugation, the Y272A 4.28 ± 0.09 68.7 ± 10.7 1.04 supernatant was subjected to a HisTrap HP column (5 mL, GE ff Y272F 4.69 ± 0.21 92.3 ± 26.0 0.85 Healthcare). The column was washed with bu er A containing 15 ff wild type 3 1.88 ± 0.01 53.6 ± 9.3 0.64 mM imidazole (30 CV), and then the protein was eluted with bu er A containing 300 mM imidazole (5 CV). The protein solution was N121A 3.86 ± 0.24 78.9 ± 20.2 0.82 a loaded on Hi-Load 16/60 Superdex 200 prep grade (GE Healthcare) R239A n.d. n.d. n.d. with gel-filtration buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, R239V n.d. n.d. n.d. and 2 mM dithiothreitol) after addition of 1 mM EDTA and rotation R239M n.d. n.d. n.d. for 1 h. The protein was concentrated to 15 mg/mL using a 10 kDa Y272A n.d. n.d. n.d. MWCO spin filter (Millipore, USA). The protein concentration was Y272F 4.78 ± 0.02 116.2 ± 18.8 0.69 measured by UV absorption at 280 nm (extinction coefficients, 0.678/ a · n.d. = not determined. M cm). Protein Crystallization and Structure Determination. The following procedure was undertaken under anaerobic conditions.13 introduction of the mutations to the residues that are predicted PEG 3350 (30% w/v), 200 mM sodium tartrate, 20% DMF, and 15 to be involved in the substrate binding, confirming the mg/mL AndA were used for crystallization, and AndA-apo crystals importance of these residues in the substrate recognition. were obtained with the sitting-drop vapor-diffusion method at 20 °C. Preandiloid B (2) or preandiloid C (3) was introduced into the AndA Additionally, the Km values were almost identical for both crystal by soaking in 30% (w/v) PEG3350, 8% tacsimate pH 7.0, 20% substrates 2 and 3, which further supports the crystallographic α observation that the two substrates bind to the enzyme in a DMF, 5 mM FeSO4,10mM KG, and 20 mM 2 or 3 for 6 h at 20 °C. AndA-apo crystal and substrate-soaked crystals were moved into very similar manner. 25% (v/v) glycerol added solution for 10 s and then frozen with liquid nitrogen. The X-ray diffraction data for AndA (BL-1A, Photon ■ CONCLUSION 14 Factory, Japan) were processed and scaled using XDS and In this study, we have provided new insight into the enzymatic AIMLESS,15 respectively. The structure of PrhA8 (PDB ID: 5YBM) synthesis of the unique bicyclo[2.2.2]octane system of was used as a search model for the molecular replacement procedure 16 17 18 andiconin (4), by crystallographic and computational studies. (Phaser in PHENIX ). Coot and Phenix.Refine were utilized for fi The crystal structures of AndA revealed the presence of the crystallographic re nement. The structural data of 2 and 3 were prepared in Chem3D Ultra software (CambridgeSoft). Substrate uncommon lid-like region that locks the substrate in the active fi site, whose importance for the rearrangement reaction was occupancy was set at 1.0. The nal crystallographic data are listed in Table S2. further validated by the mutational experiments. Furthermore, Computational Details. For all DFT calculations, the Gaussian the computational calculation based on the crystal structure of 16 program19 and/or Reaction Plus programs20 were utilized. AndA delineated a plausible reaction mechanism for the Geometry optimizations were conducted at the UB3LYP/6-31+G- bridged-ring formation by AndA. However, the mechanism for (d,p) level21 in the gas phase, without any symmetry restrictions. At the very last step of the reaction, to reduce the product radical the same level of theory, vibrational frequency calculations were 8 into 4, still remains enigmatic. Similar radical quenching to carried out to confirm that a local minimum has no imaginary complete the reaction is also required for several other αKG- frequency and each TS possesses only a single imaginary frequency. Intrinsic reaction coordinate calculations22 for all TSs were conducted dependent enzymes, such as FtmOx1 (endoperoxidase), CarC 23 (epimerase/dehydrogenase), and SnoN (epimerase), but the with GRRM11 based on Gaussian 16 (Tables S3 to S7). In this mechanisms for the radical reductions by these enzymes are study, the Gibbs free energy was adopted as the basis for discussion. Mutagenesis Analysis. Mutagenesis was performed using still unclear, despite the availability of their crystal structur- 3a,4a,b PrimeSTAR mutagenesis basal kit (Wako Pure Chemical Industries, es. Thus, further studies are required to identify the Ltd.) using full-length andA in pET28a as a template. Each primer reducing agent actually used in the “in vivo” reactions and the pair (andA-e66a-f and andA-e66a-r, andA-e66d-f and andA-e66d-r, key factors that determine whether the enzyme functions as an andA-e66q-f and andA-e66q-r, andA-n121a-f and andA-n121a-r, oxidative enzyme or an isomerase. andA-r239a-f and andA-r239a-r, andA-r239m-f and andA-r239m-r,

9748 DOI: 10.1021/jacs.8b06084 J. Am. Chem. Soc. 2018, 140, 9743−9750 Journal of the American Chemical Society Article andA-r239v-f and andA-r239v-r, andA-y272a-f and andA-y272a-r, and ■ REFERENCES andA-y272f-f and andA-y272f-r) was used for constructing E66A, E66D, E66Q, N121A, R239A, R239M, R239 V, Y272A, and Y272F, (1) (a) Tang, M.-C.; Zou, Y.; Watanabe, K.; Walsh, C. T.; Tang, Y. Chem. Rev. 2017, 117, 5226−5333. (b) Richter, M. Nat. Prod. Rep. respectively (Table S1). The plasmids were introduced into E. coli − − RosettaII(DE3)pLysS after DpnI (Wako Pure Chemical Industries, 2013, 30, 1324 1345. (c) Cox, R. J. Nat. Prod. Rep. 2014, 31, 1405 fi 1424. Ltd.) digestion. Expression and puri cation conditions were the same − as for the wild-type AndA. For the in vitro assay, 5 μM preandiloid B (2) (a) Hausinger, R. P. Crit. Rev. Biochem. Mol. Biol. 2004, 39,21 (2)or5μM preandiloid C (3) was added to 20 mM Tris-HCl (pH 68. (b) Loenarz, C.; Schofield, C. J. Nat. Chem. Biol. 2008, 4, 152. ff α (c) Matsuda, Y.; Abe, I. Nat. Prod. Rep. 2016, 33,26−53. (d) Matsuda, 7.5) bu er containing 200 mM NaCl, 0.1 mM FeSO4, 2.5 mM KG, 4 mM ascorbate, and 5 μM AndA (wild-type and mutants), in a final Y.; Awakawa, T.; Mori, T.; Abe, I. Curr. Opin. Chem. Biol. 2016, 31, − volume of 50 μL. After 1 h of enzymatic reaction at 30 °C, 50 μLof 1 7. (e) Nakamura, H.; Matsuda, Y.; Abe, I. Nat. Prod. Rep. 2018, methanol was added to each reaction mixture and then analyzed by DOI: 10.1039/C7NP00055C. LC-MS. LC-MS analysis was conducted on a Bruker Compact qTOF (3) (a) Yan, W.; Song, H.; Song, F.; Guo, Y.; Wu, C.-H.; Her, A. S.; mass spectrometer with a Shimadzu Prominence HPLC system, using Pu, Y.; Wang, S.; Naowarojna, N.; Weitz, A.; Hendrich, M. P.; electrospray ionization with a COSMOSIL 2.5C -MS-II column (2.0 Costello, C. E.; Zhang, L.; Liu, P.; Zhang, Y. J. Nature 2015, 527, 18 − ̈ i.d. × 75 mm; Nacalai Tesque, Inc.), and the separation was 539 543. (b) Brauer, A.; Beck, P.; Hintermann, L.; Groll, M. Angew. − performed using the following condition: 70% aqueous acetonitrile Chem., Int. Ed. 2016, 55, 422 426. containing 20 mM formic acid, 0.1 mL/min. To determine the (4) (a) Chang, W.-c.; Guo, Y.; Wang, C.; Butch, S. E.; Rosenzweig, − kinetics parameters of AndA and its mutants with 2 and 3 (5, 10, 50, A. C.; Boal, A. K.; Krebs, C.; Bollinger, J. M. Science 2014, 343, 1140 100, and 200 μM, duplicate), 50 μL of reaction mixture was incubated 1144. (b) Siitonen, V.; Selvaraj, B.; Niiranen, L.; Lindqvist, Y.; ̈ ̈ with 0.1 μM enzyme for 1 min at 30 °C. The reaction was stopped by Schneider, G.; Metsa-Ketela,M.Proc. Natl. Acad. Sci. U. S. A. 2016, adding 50 μL of methanol, and the intensity of consumed substrate 113, 5251−5256. (c) Matsuda, Y.; Wakimoto, T.; Mori, T.; Awakawa, was measured by LC-MS. GraphPad Prism 7 for Windows (GraphPad T.; Abe, I. J. Am. Chem. Soc. 2014, 136, 15326−15336. Software,Inc.,LaJolla,CA,USA)wasusedforKM and kcat (5) (a) Price, J. C.; Barr, E. W.; Glass, T. E.; Krebs, C.; Bollinger, J. calculation. M. J. Am. Chem. Soc. 2003, 125, 13008−13009. (b) Nam, W.; Lee, Y.- M.; Fukuzumi, S. Acc. Chem. Res. 2014, 47, 1146−1154. ■ ASSOCIATED CONTENT (6) (a) Martinez, S.; Hausinger, R. P. J. Biol. Chem. 2015, 290, − * 20702 20711. (b) Liao, H.; Li, J.; Huang, J.; Davidson, M.; Kurnikov, S Supporting Information I.; Lin, T.; Lee, J. L.; Kurnikova, M.; Guo, Y.; Chan, N.; Chang, W. The Supporting Information is available free of charge on the Angew. Chem., Int. Ed. 2018, 57, 1831−1835. ACS Publications website at DOI: 10.1021/jacs.8b06084. (7) (a) Clifton, I. J.; McDonough, M. A.; Ehrismann, D.; Kershaw, Supplementary figures, scheme, and tables (PDF) N. J.; Granatino, N.; Schofield, C. J. J. Inorg. Biochem. 2006, 100, 644−669. (b) Aik, W.; McDonough, M. A.; Thalhammer, A.; Chowdhury, R.; Schofield, C. J. Curr. Opin. Struct. 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Phys. Chem. Chem. Phys. 2017, 19, 20188 20197. equally. (12) Tantillo, D. J.; Jiangang, C.; Houk, K. N. Curr. Opin. Chem. Biol. 1998, 2, 743−750. Notes (13) Senda, M.; Kishigami, S.; Kimura, S.; Senda, T. Acta The authors declare no competing financial interest. Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2007, 63, 311−314. (14) Kabsch, W. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, ■ ACKNOWLEDGMENTS 133−144. (15) Evans, P. R.; Murshudov, G. N. Acta Crystallogr., Sect. D: Biol. This work was supported in part by a Grant-in-Aid for − fi Crystallogr. 2013, 69, 1204 1214. Scienti c Research from the Ministry of Education, Culture, (16) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. Sports, Science and Technology, Japan (JSPS KAKENHI D.; Storoni, L. C.; Read, R. J. J. Appl. Crystallogr. 2007, 40, 658−674. Grant Numbers JP15H01836, JP16H06443, JP16H06454, and (17) Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D: Biol. JP17H05430), JST/NSFC Strategic International Collabora- Crystallogr. 2004, 60, 2126−2132. tive Research Program and JSPS Research Fellowships for (18) Afonine, P. V.; Grosse-Kunstleve, R. W.; Echols, N.; Headd, J. Young Scientists (to Y.N. and T.M.), the Platform for Drug J.; Moriarty, N. W.; Mustyakimov, M.; Terwilliger, T. C.; Urzhumtsev, A.; Zwart, P. H.; Adams, P. D. Acta Crystallogr., Sect. D: Biol. Discovery, Informatics, and Structural Life Science (PDIS), − and Basis for Supporting Innovative Drug Discovery and Life Crystallogr. 2012, 68, 352 367. Science Research (BINDS) from the Japan Agency for Medical (19) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, Research and Development (AMED). The synchrotron G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; radiation experiments were performed at the BL-1A of the Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. 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