rangement in Anditomin Biosynthesis - ACS Publications - American

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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 J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06084 • Publication Date (Web): 04 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Journal of the American Chemical Society

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 1130033, 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 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 and provided insight into how AndA 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 As an excellent designer of small molecules, nature has evolved enzymes that perform diverse chemical transformations, often with the aid of a variety of cofactors and cosubstrates, thus generating the complex and intriguing frameworks of natural products.1 Non-heme iron and α-ketoglutarate (αKG)-dependent enzymes are a widespread and major class of enzymes involved in both primary and secondary metabolism.2 They are mostly engaged in simple oxidative reactions, such as hydroxylation and dehydrogenation, but some atypical reactions are catalyzed by this class of enzymes, including the endoperoxidation by FtmOx1 (FtmF) and the epoxidation by AsqJ.3 Moreover, some αKG-dependent enzymes exhibit isomerase or epimerase activity, although they can also work as oxidative enzymes.4 For the catalysis by Fe(II)/αKG-dependent enzymes, there is

a strong consensus that the oxidative decarboxylation of αKG first generates a highly active ferryl-oxo species, which then abstracts a hydrogen atom from a substrate to initiate the reaction.5 Yet, the detailed reaction mechanisms for many αKG-dependent enzymes remain poorly understood, as even the mechanism for the dehydrogenation, although it is apparently simple, is still controversial.6 Therefore, it is important to utilize several different approaches to clarify the reactions catalyzed by αKG-dependent enzymes, which will facilitate future bioengineering efforts with this class of enzymes.

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Figure 1. Structure of anditomin (1) and AndA-catalyzed two-step reactions. The genuine “reducing agent” required for the second reaction to provide andiconin (4) has yet to be identified for the in vivo reaction.

AndA, involved in the biosynthesis of the fungal meroterpenoid anditomin (1), is an αKG-dependent enzyme that catalyzes two consecutive reactions to construct a unique bridged ring system (Figure 1).4c AndA first accepts preandiloid B (2) as a substrate to introduce the C–C double bond between C1 and C2 to yield the enone preandiloid C (3), which is further utilized as the substrate for the second reaction, in which it undergoes an intriguing skeletal rearrangement to produce andiconin (4). Although the first reaction is a common dehydrogenation event, the conversion from 3 to 4 is an atypical isomerization. Our previous study revealed that the in vitro enzyme reaction of AndA requires ascorbate as an essential factor, which probably reflects the fact that AndA functions as an isomerase.4c However, the mechanisms for the synthesis of the bridged ring and how the enzyme allows for this drastic structural rearrangement have yet to be elucidated. In this study, to obtain the structural basis for the AndA-catalyzed reactions, we solved the X-ray crystal structures of AndA complexed with iron, αKG, and the individual substrates (2 and 3), which revealed the binding modes of the substrates and suggested the requirement of the ferryl-oxo species isomerization prior to the hydrogen abstraction. Further mutational studies revealed some key residues specifically important for the second reaction to produce 4. We also performed computational calculations of the rearrangement to construct the bridged-ring, and successfully provided a rationale for this conversion.

RESULTS AND DISCUSSION Crystallographic Studies on AndA. To obtain insight into how AndA participates in the oxidative rearrangement reaction, we sought to crystallize AndA for X-ray diffraction experiments. Since our initial attempt to crystallize the full length AndA was not successful, we prepared a truncated protein lacking eight residues at the Nterminus (Met1-Tyr8), which is predicted to be a disordered region. The truncated AndA was treated with EDTA prior to crystallization to completely remove any residual iron, and the metal-free enzyme was then crystallized under anaerobic conditions, leading to the X-ray crystal structure of AndA in its apo form at 2.5 Å resolu-

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tion (PDB ID: 5ZM2; Figure 2A and Table S2). The crystal structure revealed that AndA exists as a symmetric homodimer, constructed via several hydrogen bond networks at the interface of the two monomeric units (Figure 2A). This observation is consistent with the apparent molecular weight of AndA in its native state predicted by a gel filtration experiment. AndA possesses a doublestranded β-helix fold and forms a funnel-like reaction chamber conserved in the jelly-roll barrel, as commonly seen in αKG-dependent dioxygenases (Figure 2A).7 Accordingly, AndA exhibits a highly similar overall structure to other αKG-dependent enzymes involved in fungal 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 substrates 2 or 3 were investigated by soaking them into 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 to 2E, S1, 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 manner, together with the facial triad and a water molecule (w1), to form an octahedral iron complex (Figures 2D and 2E). The αKG also interacts with the His79, Gln132, Thr173, and Arg224 residues through hydrogen bonding networks (Figure 2C). The quaternary complexes including substrates (2 or 3) showed that both substrates are bound to AndA in an almost identical fashion (Figure S4 and S5, RMSD of 0.19 Å for the 263 Cα atoms), and that they are located on the opposite side of the facial triad, across from the ferrous ion (Figures 2D and 2E). As often seen in this class of enzymes, the substrate binding pocket is constructed by the engagement of both monomers of AndA: the loop between α2 and β3 (loop 58-76, Loop A) from one monomer and the loop between α7’ and α8’ (loop 261’-281’, Loop B’; hereafter the apostrophes designate residues from the other chain of that without apostrophes) from the other are adjacently located to form a binding site, together with Arg239 on another loop (Figures 2B, S6, and S7). Arg239 also interacts with the carboxyl group of Glu66. Interestingly, Loop A was only observed upon substrate binding (Figures 2A, 2B, and S6), indicating that it is highly flexible and that it serves as a lid that encapsulates the substrates in the active site of AndA. This lid-like region appears to recognize the D/E rings of the substrates, as suggested by the hydrogen bond networks observed in this area: two carbonyl oxygens (O3 and O5) both interact with the phenolic hydroxyl group of Tyr272’ as well as the backbones of Gln67 and Ile70, via one water molecule (w3 or w4), while O4 is hydrogen bonded with two water molecules (w2 and w3) that in turn interact with the carboxyl group of Glu66 and the backbones of Lys63 and Gln67 (Figures 2D, 2E, and S7). Meanwhile, the terpenoid moiety (the A/B/C rings) of the substrates is relatively loosely bound to the enzyme, since the interaction between O1


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and Asn121 is the only hydrogen bond detected in this region (Figures 2D and 2E). In line with the common reaction mechanism for αKGdependent dioxygenases, the αKG in the active site should initially undergo oxidative decarboxylation to generate the highly active ferryl-oxo species, in which the oxo group is located trans to His135. It was previously thought that this ferryl-oxo species directly abstracts a hydrogen atom from a substrate, as proposed for the well-studied αKG-dependent enzyme TauD.10 However, recent spectroscopic and computational studies indicated that the isomerization (or rotation) of the oxo group, which proceeds with a low energy barrier, is required before the hydrogen abstraction in the catalysis by several αKG-dependent enzymes.11 Thus, we sought to investigate the possibility that the isomerization event occurs in the AndA-catalyzed reactions. Although it is impossible to precisely determine the position of the oxo group, it would be close to the water molecule (w1) coordinated trans to His135 and to the carboxylic oxygen

of αKG bound trans to His213 before and after the rotation, respectively. Therefore, we measured the distances between these oxygen atoms and the carbon atoms of the substrates from which a hydrogen atom could potentially be abstracted. In the first reaction to yield the enone 3, a hydrogen atom at C1 or C2 of 2 should be abstracted at the beginning of the reaction. In the quaternary AndA/Fe/αKG/2 complex, the distances from C1 or C2 to w1 are 4.4 and 4.0 Å, respectively, while those to the carboxylic oxygen of αKG are 3.3 and 3.7 Å, respectively (Figure 2D), suggesting that the isomerization could occur to facilitate the hydrogen abstraction. For the second reaction, a hydrogen atom at C12 is initially abstracted,5c and the oxo rotation seems to be more important than that in the first reaction, since the carboxylic oxygen of αKG is located much closer to C12 than w1 (3.5 vs. 4.9 Å) in the quaternary AndA/Fe/αKG/3 complex (Figure 2E). Collectively, our crystallographic study suggests the requirement of the oxo group isomerization, but this should be confirmed by future studies.



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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 αKGbinding 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).

On the basis of the above discussion, the α-oriented hydrogen at C1 would initially be abstracted in the dehydrogenation reaction to afford 3. To complete the desaturation, the vicinal hydrogen at C2 could be abstracted, followed by a diradical recombination to introduce the C– C double bond (Figure S8A). However, a recent mechanistic study of the AsqJ-catalyzed desaturation indicated alternative mechanisms for this type of reaction: after the

generation of the substrate radical, it could then undergo (i) the oxygen rebound and following dehydration, or (ii) the oxidization by Fe(III) to a cationic species and subsequent deprotonation, to install the double bond.6b At this moment, none of the three possibilities could be ruled out, but given the structural similarity between AndA and AsqJ, AndA could also use the same strategy for the desaturation as that of AsqJ (Figure S8B).


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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).

Computational Calculations of the AndA-Catalyzed Rearrangement. We then performed a density functional theory (DFT) calculation (UB3LYP/6–31 + G(d,p)) to obtain deeper insight into the reaction pathway and the mechanism for the rearrangement to provide 4. First, we confirmed that the 3D-structure and conformation of the substrate 3 used for the calculation are essentially the same as those of 3 in the co-crystal (Table S3 and Figure S9). After the initial hydrogen abstraction from C12, we identified two possible multistep reaction pathways (routes A and B) for the dramatic skeletal rearrangement from 5 to 8, including (i) C8–O2 bond cleavage, (ii) C12– C5’ bond formation, and (iii) C8–C2’ bond formation, accompanied by several conformational changes (Figures 3 and S10 and Tables S4 and S5). The energy diagram suggested that both routes are thermodynamically and kinetically favorable: (i) the activation barriers are all low enough for the reactions to proceed smoothly at ambient temperature, (ii) the entire energy profile descends as the reactions proceed, and (iii) the overall exergonicity is very large (∆G = –19.8 kcal/mol). Notably, the geometrical features of the computed 5 are in good agreement with those of the experimentally obtained 3 cocrystallized with AndA. We found that the intermediate 7a and the subsequent cascade reactions to afford 8 had identical geometry and energy in both routes. However,

the key intermediate 7a can be reached from the reactant 5 via two different pathways. In route A, the C8–O2 bond cleavage occurs prior to the C12–C5’ bond formation, via the transition state (TS) TS_5-6 to give the exo-methylene 6 with a large stabilization energy (∆G = –19.4 kcal/mol), due to an intrinsically stable tertiary carbon radical. The activation energy for the C–O bond cleavage is only 6.6 kcal/mol. Conversely, route B starts with 5-endo-trig (anti-Baldwin) cyclization to produce the α-oxy carbon radical intermediate 6’, with an activation barrier of 23.4 kcal/mol. 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 (∆G = –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 (Figures 2D and 2E). To further increase the reliability of the aboveproposed 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 rearrangement 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 Characterization of AndA. To investigate how AndA is engaged in the rearrangement and to examine the importance of the amino acid residues comprising the active site, we performed mutational experiments on selected residues with side chains involved in the substrate binding; namely, Glu66, Asn121, Arg239, and Tyr272’. First, the point mutation of Asn121 (N121A) resulted in reduced activity, but did not completely abolish the conversions (Figure 4A), suggesting that the hydrogen bonding between Asn121 and O1 is not crucial for the reactions to proceed. Among the residues in the lid-like region, the mutations of Glu66 (E66A, E66D, and E66Q) only yielded insoluble proteins, which hampered the evaluation of the importance of this residue in the catalysis. Nevertheless, the mutageneses of Arg239 and Tyr272’ provided intriguing outcomes. The Y272F mutant exhibited activity comparable to that of the wild type enzyme, but all of the other constructed mutants, R239A, R239M, R239V, and Y272A, only produced the dehydrogenated product 3 and lost the ability to synthesize 4 when 2 was used as a substrate (Figure 4A). Consistently, 3 was not further transformed into 4 when the above-mentioned four mutants were used for the reaction (Figure 4B). In contrast to the first desaturation reaction, which proceeds relatively inside the substrate binding pocket, the second reaction occurs near the entrance of the pocket and also requires a significant structural rearrangement. Since both Arg239 and Tyr272’ interact with the lid-like region, the abolishment of the second reaction after the introduction of mutations to these residues could be attributed to the destruction of the lid function and the exposure of the D/E rings, which undergo significant structural rearrangements, to the external environment. Collectively, AndA could play a crucial role to protect the highly reactive radical intermediates by housing them in the closed active site, thus allowing the intriguing skeletal

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rearrangement to proceed without quenching during the transformation.

Figure 4. LC-MS profiles of the products from in vitro enzymatic reactions of the AndA mutants with (A) 2 or (B) 3. The chromatograms were extracted at m/z 413.233 (for 2; blue trace) and 411.217 (for 3 and 4; red trace). WT = wild type AndA. NC = negative control.

Finally, we performed biochemical characterization of AndA and its mutants. Consistent with our previous in vitro enzymatic reaction with the wild type AndA,4c the reactions by the mutants that produce 4 required ascorbate as an essential factor (Figure S12), which could be attributed to the prediction that ascorbate is utilized to finalize the reaction. Interestingly, ascorbate is absolutely essential even for the mutants that are only able to perform the first-round reaction to produce 3 (Figure S12), although the dehydrogenation reaction does not apparently require a reducing agent. This observation that ascorbate is indispensable for all the chemistry by AndA indicates that ascorbate has more function(s) than just reducing the product radical. One possible explanation would be that the productions of 3 and 4 are significantly uncoupled from the αKG oxidation and that ascor-


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bate is inevitably required to maintain the oxidation state of iron, but this needs to be further clarified in the future studies. We also measured the kinetic parameters for AndA and its mutants (Table 1). The Km values for both 2 and 3 increased after the introduction of the mutations to the residues that are predicted to be involved in the substrate binding, confirming the importance of these residues in the substrate recognition. Additionally, the Km values were almost identical for both substrates 2 and 3, which further supports the crystallographic observation that the two substrates bind to the enzyme in a very similar manner. Table 1. Steady-state kinetic parameters for AndA and its mutants. -1

protein substrate kcat (sec ) wild type N121A R239A R239V R239M Y272A Y272F wild type N121A R239A R239V R239M Y272A

2

3

Y272F

-1

KM (µM)

-

kcat/KM (sec µM 1 )

6.31 ± 0.14 52.6 ± 9.7

2.00

5.00 ± 0.10 2.13 ± 0.11 3.09 ± 0.12 3.05 ± 0.07 4.28 ± 0.09 4.69 ± 0.21

84.0 ± 10.7 70.1 ± 28.0 75.8 ± 22.3 85.5 ± 14.0 68.7 ± 10.7 92.3 ± 26.0

1.00 0.48 0.68 0.59 1.04 0.85

1.88 ± 0.01 53.6 ± 9.3

0.64

3.86 ± 0.24 78.9 ± 20.2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 116.2 ± 4.78 ± 0.02 18.8

0.82 n.d. n.d. n.d. n.d. 0.69

n.d. = not determined

CONCLUSION In this study, we have provided new insight into the enzymatic synthesis of the unique bicyclo[2.2.2]octane system of andiconin (4), by crystallographic and computational studies. The crystal structures of AndA revealed the presence of the uncommon lid-like region that locks the substrate in the active site, whose importance for the rearrangement reaction was further validated by the mutational experiments. Furthermore, the computational calculation based on the crystal structure of AndA delineated a plausible reaction mechanism for the bridgedring formation by AndA. However, the mechanism for the very last step of the reaction, to reduce the product radical 8 into 4, still remains enigmatic. Similar radical quenching to complete the reaction is also required for several other αKG-dependent enzymes, such as FtmOx1 (endoperoxidase), CarC (epimerase/dehydrogenase), and SnoN (epimerase), but the mechanisms for the radical reductions by these enzymes are still unclear, despite the availability of their crystal structures.3a,4a,4b Thus, further studies are required to identify the reducing agent actually used in the “in vivo” reactions and the key factors that determine whether the enzyme functions as an oxidative enzyme or an isomerase.

MATERIALS AND METHODS General. Solvent and chemicals were purchased from Hampton Research (CA, USA) and Wako Chemicals Ltd. (Tokyo, Japan), unless noted otherwise. Preandiloid B (2) and preandiloid C (3) were prepared according to the previous work.4c Oligonucleotide primers were purchased from Eurofins Genetics (Tokyo, Japan). Sequence analyses were performed by Eurofins Genetics. Enzyme Expression and Purification for Crystallization. The N-terminal truncated andA gene was PCR-amplified from the previously prepared full-length andA-pET28a,4c with the primer pairs, andA-9-293-f and andA-9-293-r (Table S1). The DNA-fragments were digested with NdeI and BamHI and ligated into pET28a using T4 DNA ligase (Takara Inc.). Constructed plasmids were introduced into Escherichia coli RosettaII(DE3)pLysS (Novagen) and the transformants were cultured with Luria-Bertani medium supplemented with 12.5 µg/ml chloramphenicol and 25 µg/ml kanamycin at 37 °C to an OD600 of 0.6. The cultures were further incubated at 16 °C for 18 hr after 0.3 mM Isopropyl β-D-1-thiogalactopyranoside addition. The cells were pelleted by a 20 min centrifugation at 6,000 g and lysed by sonication in buffer A (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 5% (v/v) glycerol, and 5 mM imidazole). After the centrifugation, the supernatant was subjected to a HisTrap HP column (5 ml, GE Healthcare). The column was washed with buffer A containing 15 mM imidazole (30 CV), and then the protein was eluted with buffer A containing 300 mM imidazole (5 CV). The protein solution was loaded on Hi-LoadTM 16/60 SuperdexTM 200 prep grade (GE Healthcare) with gel-filtration buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2 mM dithiothreitol) after addition of 1 mM EDTA and rotation for 1 hr. The protein was concentrated to 15 mg/ml using a 10 kDa MWCO spin filter (Millipore, USA). The protein concentration was measured by UV absorption at 280 nm (extinction coefficients, 0.678 /Mcm). Protein Crystallization and Structure Determination. The following procedure was undertaken under anaerobic conditions.13 30% (w/v) PEG 3350, 200 mM sodium tartrate, 20% DMF, and 15 mg/ml AndA was used for crystallization, and AndA-apo crystals were obtained with the sitting-drop vapor-diffusion method at 20 °C. Preandiloid B (2) or preandiloid C (3) were introduced into the AndA crystal by soaking in 30% (w/v) PEG3350, 8% tacsimate pH 7.0, 20% DMF, 5 mM FeSO4, 10 mM αKG, and 20 mM 2 or 3 for 6 hours at 20 °C. AndA-apo crystal and substrate soaked crystals were moved into 25% (v/v) glycerol added solution for 10 sec and then frozen with liquid nitrogen. The X-ray diffraction data for AndA (BL1A, Photon Factory, Japan) were processed and scaled using XDS14 and AIMLESS,15 respectively. The structure of PrhA8 (PDB ID: 5YMB) was used as a search model for the molecular replacement procedure (Phaser in PHENIX16). Coot17 and Phenix.Refine18 was utilized for crystallographic refinement. The structural data of 2 and 3 were prepared in Chem3D Ultra software (CambridgeSoft). Substrate occupancy was set at 1.0. The final crystallographic data are listed in Table S2. Computational Details. For all DFT calculations, the Gaussian 16 program19 and/or Reaction Plus programs20 were utilized. Geometry optimizations were conducted at



the UB3LYP/6-31+G(d,p) level21 in the gas phase, without any symmetry restrictions. At the same level of theory, vibrational frequency calculations were carried out to confirm that a local minimum has no imaginary frequency and each TS possesses only a single imaginary frequency. Intrinsic reaction coordinate calculations22 for all TSs were conducted with GRRM1123 based on Gaussian 16 (Tables S3 to S7). In this study, the Gibbs free energy was adopted as the basis for discussion. Mutagenesis Analysis. Mutagenesis was performed using PrimeSTAR® Mutagenesis Basal Kit (Takara Inc.) using full-length andA in pET28a as a template. Each primer pairs (andA-e66a-f and andA-e66a-r, andA-e66d-f and andA-e66d-r, andA-e66q-f and andA-e66q-r, andAn121a-f and andA-n121a-r, andA-r239a-f and andAr239a-r, andA-r239m-f and andA-r239m-r, andA-r239v-f and andA-r239v-r, andA-y272a-f and andA-y272a-r, and andA-y272f-f and andA-y272f-r) was used for constructing E66A, E66D, E66Q, N121A, R239A, R239M, R239V, Y272A, and Y272F respectively (Table S1). The plasmids were introduced into E. coli RosettaII(DE3)pLysS after DpnI (Takara Inc.) digestion. Expression and purification condition was the same as the wild type AndA. For the in vitro assay, 5 µM preandiloid B (2) or 5 µM preandiloid C (3) was added to 20 mM Tris-HCl (pH 7.5) buffer containing 200 mM NaCl, 0.1 mM FeSO4, 2.5 mM αKG, 4 mM ascorbate, and 5 µM AndA (wild type and mutants), in a final volume of 50 µL. After 1 hr of enzymatic reaction at 30 °C, 50 µL methanol was added to each reaction mixture and then analyzed by LC-MS. LCMS analysis was conducted on a Bruker Compact qTOF mass spectrometer with a Shimadzu Prominence HPLC system, using electrospray ionization with a COSMOSIL 2.5C18-MS-II column (2.0 i.d. x 75 mm; Nacalai Tesque, Inc.), and the separation was performed using the following condition: 70% aqueous acetonitrile containing 20 mM formic acid, 0.1 ml/min. To determine the kinetics parameters of AndA and its mutants with 2 and 3 (5, 10, 50, 100, and 200 µM, duplicate), 50 µL reaction mixture was incubated with 0.1 µM enzyme for 1 min at 30 °C. The reaction was stopped by adding 50 µL methanol and the intensity of consumed substrate was measured by LC-MS. GraphPad Prism 7 for Windows (GraphPad Software, Inc., La Jolla, CA) was used for KM and kcat calculation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Supplementary Figures, Scheme, and Tables (PDF)

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected]

Author Contributions

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‡These authors contributed equally.

Notes The authors declare no competing interest.

ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant Numbers JP15H01836, JP16H06443, JP16H06454, and JP17H05430), JST/NSFC Strategic International Collaborative Research Program, and JSPS Research Fellowships for Young Scientists (to Y.N. and T.M.), the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS), and Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from Japan Agency for Medical Research and Development (AMED). The synchrotron radiation experiments were performed at the BL-1A of the Photon Factory with proposal No. 2014T006 and 2015G530. Allotment of computational resource (Project G18008) from HOKUSAI GreatWave (RIKEN) is gratefully acknowledged.

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