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Volume 591, Issue 20 p. 3391-3401
Research Letter
Free Access

Structural dissection of Shewanella oneidensis old yellow enzyme 4 bound to a Meisenheimer complex and (nitro)phenolic ligands

Jonathan Elegheert

Corresponding Author

Jonathan Elegheert

Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, Belgium

Correspondence

J. Elegheert and S. N. Savvides, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium

Tel: +44(0)1865287551 (JE)

Fax: +32(0)92217673 (SNS)

Tel: +32(0)93313660 (SNS)

E-mails: [email protected] (JE); [email protected] (SNS)

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Ann Brigé

Ann Brigé

Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, Belgium

Ablynx NV, Zwijnaarde, Belgium

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Jozef Van Beeumen

Jozef Van Beeumen

Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, Belgium

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Savvas N. Savvides

Corresponding Author

Savvas N. Savvides

Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, Belgium

VIB-UGent Center for Inflammation Research (IRC), Ghent University, Zwijnaarde, Belgium

Correspondence

J. Elegheert and S. N. Savvides, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Department of Biochemistry and Microbiology, Ghent University, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium

Tel: +44(0)1865287551 (JE)

Fax: +32(0)92217673 (SNS)

Tel: +32(0)93313660 (SNS)

E-mails: [email protected] (JE); [email protected] (SNS)

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First published: 04 September 2017
Citations: 9
Edited by Stuart Ferguson

Abstract

Shewanella oneidensis, a Gram-negative γ-proteobacterium with an extensive redox capacity, possesses four old yellow enzyme (OYE) homologs. Of these, Shewanella yellow enzyme 4 (SYE4) is implicated in resistance to oxidative stress. Here, we present a series of high-resolution crystal structures for SYE4 in the oxidized and reduced states, and in complex with phenolic ligands and the nitro-aromatic explosive picric acid. The structures unmask new features, including the identification of a binding platform for long-chain hydrophobic molecules. Furthermore, we present the first structural observation of a hydride-Meisenheimer complex of picric acid with a flavoenzyme. Overall, our study exposes the binding promiscuity of SYE4 toward a variety of electrophilic substrates and is consistent with a general detoxification function for SYE4.

Abbreviations

ADP, atomic displacement parameter

FMN, flavin mononucleotide

OYE, old yellow enzyme

PDB, Protein Data Bank

p-HBA, para-hydroxybenzaldehyde

p-MOP, para-methoxyphenol

p-MP, para-methylphenol

PUFA, poly-unsaturated fatty acid

SYE, Shewanella yellow enzyme

TNP, 2,4,6-trinitrophenol

TNT, trinitrotoluene

Since the discovery of old yellow enzyme (OYE) in the 1930s as the first flavin-containing enzyme, OYE has emerged as the archetype of the continuously growing family of OYE homologs in yeasts [1], plants [2], parasites [3], and numerous bacterial species [4-6]. OYE family enzymes are united via their capacity to bind the cofactor flavin mononucleotide (FMN) [7] noncovalently in their active site and to form charge-transfer complexes with a variety of phenolic ligands resulting in long-wavelength optical transitions. This involves a stacking interaction between the phenolic ring and the isoalloxazine ring of FMN, and hydrogen bonding of the phenol oxygen to conserved histidine/asparagine or histidine/histidine pair of residues in the active site of OYE family proteins. Furthermore, OYE family enzymes are able to reduce simple and complex unsaturated aldehydes and ketones, nitro-esters, and nitro-aromatic substrates through a ping-pong mechanism consisting of an oxidative and reductive half-reaction using the coenzyme NAD(P)H [7]. Interestingly, several OYEs were found to transform the explosives trinitrotoluene (TNT) and picric acid (2,4,6-trinitrophenol; TNP), which has triggered interest in using OYE in bioremediation processes, e.g., the phytoremediation of such recalcitrant pollutants [8].

Several studies have postulated a role for OYEs as antioxidants in different stress conditions. For example, OYE proteins play a central role in the oxidative stress response and cytoskeletal dynamics in yeast [9]. In Bacillus subtilis, genes known to be induced by oxidative stress are also upregulated under acid stress conditions, including namA that encodes the OYE family member yqjM [10]. A number of other observations also argue against a single physiological role for OYE enzymes. The genome of Shewanella oneidensis for instance encodes four OYE homologs that have been termed Shewanella yellow enzyme 1–4 (SYE1–4) [11]. A comparative study revealed a divergence in the biochemical properties of these SYE proteins; SYE4 has a preference for NADPH over NADH (SYE1–3) as the physiological reductant in the reductive half-reaction, and shows higher catalytic efficiencies toward a broader set of substrates [11]. Furthermore, SYE4 is the only of the four SYEs that was robustly induced by oxidative stress conditions [11]. A comparable variation has also been observed among the six OYE homologs in Pseudomonas putida KT2440 [12].

Here, we report high-resolution crystal structures of SYE4 in the oxidized and reduced state, and in complex with phenolic ligands para-hydroxybenzaldehyde (p-HBA), para-methoxyphenol (p-MOP), and para-methylphenol (p-MP), as well as with the nitro-aromatic explosive picric acid (TNP). Importantly, we provide unprecedented structural snapshots at high resolution of the complex of the Meisenheimer complex of TNP with SYE4. These structures (a) show in great detail how SYE4 accommodates a variety of electrophilic substrates in its active site facilitated by the plasticity of the isoalloxazine ring of the flavin cofactor, (b) reveal a possible route for the entry of candidate long-chain aliphatic substrates, and (c) together suggest a role for SYE4 in general cellular detoxification.

Materials and methods

Protein expression and purification

Recombinant SYE4 was expressed in Escherichia coli, purified to milligram quantities and crystallized as previously described [13].

Crystallization, data collection, and structure determination

Crystallization of SYE4 and preparation of SYE4–ligand complexes were performed as previously described [13]. Briefly, for crystal soaking experiments, crystals of SYE4 were washed with crystal stabilization buffer (1.6 m sodium citrate tribasic dihydrate, pH 6.3) and subsequently incubated in the same buffer supplemented with 20 mmp-HBA, p-MOP, p-MP, or TNP. Progress of complex formation with phenolic ligands was monitored by the change of crystal color from yellow to lime-green indicating establishment of charge-transfer complexes [14]. To trap a hydride-Meisenheimer complex of TNP in crystals of SYE4, a two-step procedure was adopted. First, SYE4 crystals were chemically reduced following a brief (2 min) incubation in stabilization buffer supplemented with 1 mm sodium borohydride (NaBH4). Progress of the chemical reduction was monitored by the change of the golden yellow crystals of oxidized SYE4 to colorless. In the second step, reduced crystals were incubated overnight in crystal stabilization solution (1.6 m sodium citrate tribasic dihydrate, pH 6.3) containing 20 mm TNP. A very pronounced color change from colorless to deep orange indicated that the long-lived hydride-Meisenheimer complex (H-TNP) was likely formed at high occupancy [15, 16]. As a last step, ligand-soaked crystals were transferred to the stabilization solution with added 20% (v/v) glycerol before cryo-cooling in liquid nitrogen. Diffraction data were collected under cryogenic temperatures (100 K nitrogen gas stream) at DESY-EMBL beam lines X11 and X13 and SLS beam line X06SA, and were processed and scaled using XDS/XSCALE [17].

The structure of SYE4 was determined by molecular replacement (MR) using maximum-likelihood methods implemented in the program phaser [18], using a search model generated from the coordinates of SYE1 [Protein Data Bank (PDB) entry 2GOU] that exhibits 42% sequence identity to SYE4 [19]. In the search model, nonconserved residues were replaced by alanine or glycine, while all insertions, water molecules, and the FMN cofactor were omitted. Initial model building was carried out with ARP/wARP 7.0.1 [20], with native data truncated to 1.50 Å resolution. All structures were refined with the PHENIX suite [21], using automated X-ray and B-factor weight optimization. We increased the value of the estimated standard deviation (ESD) of the FMN isoalloxazine planarity restraints from 0.020 to 0.200 in the FMN restraints file (CIF; Crystallographic Information File) to accommodate the increased butterfly bending of the reduced isoalloxazine ring during refinement and to allow movement of the atoms into the centroid of the density.

For the oxidized and p-HBA-soaked datasets, validation of the anisotropic atomic displacement parameter (ADP) model was carried out using the PARVATI server [22]. Manual model building was performed using Coot [23]. All validation was performed within the PHENIX suite [21]. Molecular representations were prepared using the program pymol [24].

Results and Discussion

Overall structure of SYE4

The crystal structure of oxidized SYE4 was refined to 1.10 Å resolution (Table 1). Consistent with its classification as an OYE homolog, SYE4 displays a α8β8-barrel topology containing a noncovalently bound FMN molecule at the core of the barrel (Fig. 1A). Besides the eight α-helices and β-strands, other features make up a typical OYE barrel. In SYE4, these extra-barrel elements include: (a) an N-terminal disordered seven-residue segment, (b) a β-hairpin that closes the bottom of the barrel, (c) a big excursion between β3 and α3 that makes up the capping subdomain of the barrel, (d) an α-helix linking β4 and α4, which in the OYE homolog OPR1 is thought to aid in substrate recognition [25], and (e) an α-helix between β8 and α8 that contributes to the binding of FMN. Thus far, three different structural arrangements have been observed for capping subdomain: an α-β-α-β-motif, a β-sheet composed of two 2-stranded β-sheets, and a two-stranded β-sheet connected by a hairpin loop and preceded by a very short α-helix. In SYE4, the capping domain is a small β-sheet involving four residues of two parallel β-strands (Fig. 1A).

Table 1. X-ray data collection and refinement statistics. Numbers in parentheses refer to the highest resolution shell. R.m.s.d., root-mean-square deviation from ideal geometry; Rmeas, multiplicity-corrected merging R-factor [39]; VM, Matthews coefficient
SYE4 oxidized SYE4 p-hydroxy-benzaldehyde SYE4 p-methoxy-phenol SYE4 p-methylphenol SYE4 TNP SYE4 reduced SYE4 TNP hydride-Meisenheimer complex
Data collection
Source

SLS

X06SA

DESY-EMBL

X13

DESY-EMBL

X11

DESY-EMBL

X11

DESY-EMBL

X11

DESY-EMBL

X11

DESY-EMBL

X11

Wavelength λ (Å) 0.8500 0.8076 0.8148 0.8148 0.8148 0.8148 0.8148
Detector Pilatus-6M MAR165 MAR165 MAR555 MAR555 MAR555 MAR555
Resolution (Å) 50.00–1.10 (1.20–1.10) 20.00–1.30 (1.35–1.30) 20.00–1.50 (1.60–1.50) 25.00–1.55 (1.59–1.55) 25.00–1.60 (1.64–1.60) 25.00–1.45 (1.49–1.45) 25.00–1.55 (1.59–1.55)
Space group P212121 P212121 P212121 P212121 P212121 P212121 P212121
Unit cell parameters (Å)
a 52.23 52.09 52.18 52.26 52.18 52.22 50.43
b 54.81 54.66 54.83 54.79 54.77 54.78 54.47
c 103.55 103.75 103.48 103.67 103.50 103.23 105.27
VM3·Da−1) 1.90 1.89 1.89 1.90 1.89 1.89 1.85
Unique reflections 120 389 (27 478) 71 727 (6642) 47 629 (8287) 42 707 (3064) 39 489 (2829) 52 643 (3809) 42 455 (2977)
Multiplicity 4.5 (4.4) 5.3 (4.9) 5.2 (4.1) 4.5 (2.4) 6.9 (3.7) 7.2 (7.0) 5.4 (5.3)
Completeness (%) 99.4 (99.8) 97.5 (85.5) 98.5 (98.9) 97.1 (95.4) 98.9 (96.8) 98.8 (98.2) 99.3 (95.9)
Rmeas (%) 4.6 (34.2) 11.8 (66.7) 8.0 (43.4) 7.0 (54.0) 7.1 (49.4) 6.3 (58.5) 8.8 (60.8)
Average I/σ(I) 18.3 (5.0) 9.7 (2.7) 18.4 (3.6) 21.8 (2.7) 24.6 (3.8) 29.1 (4.1) 19.9 (3.0)
Refinement
PDB codes 4B5N 5K1K 5K1M 5K1Q 5K1W 5K0R 5K1U
Resolution (Å) 50.00–1.10 (1.14–1.10) 20.00–1.30 (1.33–1.30) 20.00–1.50 (1.54–1.50) 25.00–1.55 (1.59–1.55) 25.00–1.60 (1.64–1.60) 25.00–1.45 (1.49–1.45) 25.00–1.55 (1.59–1.55)
Reflections (working set/test set) 118 977/1411 69 712/2014 45 653/1975 40 633/2073 37 564/1925 50 691/1945 40 391/2063
Rwork/Rfree 0.1270/0.1405 (0.1418/0.1766) 0.1217/0.1480 (0.1998/0.2322) 0.1424/0.1736 (0.2267/0.2744) 0.1365/0.1717 (0.2750/0.3116) 0.1383/0.1695 (0.2206/0.2499) 0.1480/0.1790 (0.2421/0.2772) 0.1303/0.1657 (0.2218/0.2467)
R.m.s. deviations
Bonds (Å) 0.009 0.010 0.005 0.008 0.005 0.005 0.006
Angles (°) 1.426 1.360 1.053 1.176 1.029 1.065 1.086
Average ADP values (Å2)
Protein 12.35 12.13 11.18 11.50 12.53 14.13 10.14
Ligands 9.50 22.83 14.12 16.09 17.39 13.54 13.33
Waters 24.35 32.61 27.18 30.17 27.00 29.35 25.18
Details are in the caption following the image
Structure of SYE4 and identification of a binding platform for long aliphatic chains. (A) Overall structure of oxidized SYE4. The FMN cofactor and putative n-octadecane are shown in ball-and-stick representation. mFo-DFc density, contoured at 3.0 σ, is shown as a green mesh. (B) Surface representation of SYE4, showing the tunnel giving access to the active site. For comparison, we superposed SYE1 [19] and SYE4 (0.552 Å root-mean-square deviation over 214 Cα-atoms); SYE1 loop 6, with Trp274 at its tip, folds toward the center of the barrel and contributes significantly to the lining of the tunnel, making the entrance to the active site comparatively narrow. (C) Aliphatic and aromatic SYE4 residues provide a hydrophobic binding platform for long-chain aliphatic molecules. 2mFo-DFc density, contoured at 1.0 σ, is shown as a blue mesh. The final model for a putative n-octadecane is shown in ball-and-stick representation. Electron density maps were calculated with the PHENIX suite [21]. The analysis of protein hydrophobicity, color-annotated onto the SYE4 structure, was based on the Eisenberg hydrophobicity scale [40].

The SYE4 active site architecture

The active site of SYE4 is composed of a broad tunnel about 15 Å long and is lined by aromatic and hydrophobic residues. Pronounced differences between SYE1 and SYE4 are found at the opening of the substrate-binding cavity. Loop 6 of SYE1 folds toward the center of the barrel and Trp274, centered at the tip of this loop, contributes to a significant part of the lining of the tunnel, making the entrance to the active site very narrow. The corresponding loop in SYE4 is not shorter but folds back at its C-terminal end toward α5, effectively broadening the substrate channel (Fig. 1B). This observation provides an explanation for the higher binding capacity of SYE4 for ‘bulkier’ electrophilic substrates [11].

Flavin mononucleotide is tightly bound in the protein; the amino acids that contact the riboflavin moiety of FMN are conserved among the known OYE structures: the flavin N5 interacts with the main chain amide of Thr35, O4 is hydrogen-bonded to the Oγ of Thr35 and the main chain amide of Ala66, and N3 and O2 interact with the side chain of Gln108. O2 makes an additional hydrogen bond with the side chain amine of His181 and the guanidinium group of Arg234.

The two hydroxyl groups of the ribityl moiety most proximal to the isoalloxazine ring are each hydrogen-bonded to the guanidinium group of Arg234, and to the main chain carbonyl oxygen of Pro33 and a water molecule, respectively. The third hydroxyl group and the phosphate oxygens are involved in an extensive hydrogen-bonding network involving the side chain of Arg325, the main chain amides of this arginine, Gly302 and Ser303, and three water molecules. In contrast to the si-face of the flavin, the ribityl side chain, and the dimethylbenzene ring, which are all solvent-accessible, the re-face of the flavin is buried in the structure and is surrounded by residues from Loop 1 (Fig. 1A).

Identification of a binding platform for long aliphatic substrates

During refinement of the crystal structure of SYE4 in its oxidized form, difference electron density maps provided clear evidence for the binding of a long, linear molecule near the active site entrance (Fig. 1A). We modeled this molecule as a nonbranched alkane chain (C18: n-octadecane), based on (a) apparent bond lengths and conformational flexibility of the chain, (b) hydrophobic protein environment, and (c) apparent absence of functional groups. Despite the high overall resolution of the dataset, the ‘head’ and ‘tail’ of the molecule make fewer contacts with SYE4 and were consequently less well resolved in the electron density, which prevented unequivocal interpretation of the corresponding chemical identity. The ‘head’ extends into the entrance of the active site pocket but not beyond, and there was no evidence for stacking of the molecule against the FMN isoalloxazine ring or for hydrogen bonding of the molecule to the catalytic His/Asn pair. Contacts between the molecule and SYE4 are based on Van der Waals (VdW) interactions with residues Tyr240, Phe241, Ile272, Phe273, Ile277, and Phe279 that form the hydrophobic lining of the active site environment. We propose that this observation uncovers a probable route on the enzyme surface through which longer chain aliphatic substrates can be guided toward the active site (Fig. 1C).

SYE4 catalyzes the reduction of a variety of α,β-unsaturated aldehydes, such as the lipid peroxidation product acrolein [11]. Also, from the four SYEs in S. oneidensis, SYE4 is the only one upregulated under conditions of oxidative stress [11]. Combined with the apparent presence of a long-chain alkane in close proximity to the active site gorge, we propose that SYE4 plays a physiological role in the cellular oxidative stress response. A number of observations corroborate this hypothesis. Straight chain α,β-unsaturated aldehydes are degradation products of lipid oxidation processes (oxidative damage). Polyunsaturated fatty acids (PUFAs), mainly restricted to eukaryotes, are more prone to attack by reactive oxygen species. Yet, some prokaryotes have been shown to increase fatty acid desaturation in cellular response to environmental stress [26]. Also, bacterial strains from low-temperature and high-pressure marine environments appear to synthesize PUFAs, such as linoleic acid (C18:2n-6), or, more generally, n-3 long-chain PUFAs [27, 28]. This is most interesting, as one of the strains identified in these studies was Photobacterium profundum, which contains two OYE homologs with a sequence identity of 71% and 78% to SYE2 and SYE4, respectively. Also, Vibrio T3615 and Shewanella sp. GA-22 were among the other strains identified [27, 28]. OYE homologs have been found in close relatives of both strains. Lipid peroxidation, initiated in E. coli by the addition of oxidizing agents to linoleic acid has been shown to result in the induction of the OYE homolog N-ethylmaleimide reductase [29]. Testament to their toxic roles in a cellular environment, PUFAs and their lipid oxidation products can contribute significantly to the oxidative damage of proteins as occurs under conditions of oxidative stress [30].

Enzymes involved in detoxification pathways are typically marked by wide species differences, several isoforms displaying differing kinetics, broad specificity, and high catalytic efficiency; these are all criteria that are met by the SYE proteins and SYE4 in particular [11].

Structure of SYE4 in complex with phenolic ligands

The active site environment of the OYEs has traditionally been probed with phenolic ligands, small molecules that make extensive use of the hydrogen-bonding capacity of the active site and with which OYEs form so called ‘charge transfer’ complexes. Within the SYEs, SYE4 is the only enzyme capable of forming long-wavelength charge-transfer complexes with phenolic ligands with pKa values ranging from 5.4 to 10.3 [11].

We determined crystal structures of oxidized SYE4 in complex with the phenolic ligands p-HBA (1.30 Å), p-MOP (1.50 Å), and p-MP (1.55 Å; Fig. 2A and Table 1).

Details are in the caption following the image
Structures of SYE4 in complex with phenolic ligands. (A) Chemical structure of the anionic form of the phenolic ligands p-HBA, p-MOP, and p-MP. (B–D) Binding modes of (B) p-HBA, (C) p-MOP, and (D) p-MP in the SYE4 active site. All three phenolic ligands display canonical hydrogen bonding to the Asn185/His182 catalytic pair through their phenolate oxygen. p-MOP shows a discretely disordered stacking on top of the FMN isoalloxazine ring; the separate conformers are colored yellow and salmon pink, respectively. 2mFo-DFc density, contoured at 1.0 σ, is shown as a blue mesh. mFo-DFc density, contoured at + 3.0 σ or − 3.0 σ, is shown as a green or red mesh, respectively. Electron density maps were calculated with the PHENIX suite [21].

In OYEs, Tyr375 (OYE1 numbering) is the most recurrent hydrogen bond donor for the para-substituent and is conserved in all plant and yeast OYEs except for Schizosaccharomyces pombe. In SYE1, Trp274, centered at the tip of the loop 6, performs this function. In SYE4 however, both equivalent residues are absent, meaning that the para-substituent is not immobilized by hydrogen bonding (Fig 2B,C,D). This also explains why in the structure of SYE4 complexed with p-MOP, p-MOP shows a disordered stacking on top of the FMN isoalloxazine ring (Fig. 2C).

Conformation of FMN in oxidized and reduced SYE4

The tricyclic isoalloxazine ring of the FMN cofactor in oxidized SYE4 shows only mild ‘butterfly bending’ around the N5–N10 axis (Fig. 3B). This is in agreement with our hypothesis that a planar isoalloxazine ring will be observed when a Leu (Leu34 in SYE4) is positioned at the re-face of the FMN dimethylbenzene moiety and a bent ring when this position is occupied by a Met [19], as is the case in the structures of e.g., SYE1 [19], YqjM [31], AtOPR3 [32], and LeOPR3 [2]. The dense crystal packing of the SYE4 crystals did not allow soaking of the relatively bulky NADH; chemical reduction was achieved by soaking crystals of oxidized SYE4 in a mother liquor solution containing NaBH4 (Fig. 3A). Analysis of the structure shows that reduction of the SYE4 FMN to FMNH2 results in an increased bending of the isoalloxazine ring about the N5–N10 axis, as is previously observed in OYE homologs (Fig. 3B).

Details are in the caption following the image
Conformational plasticity of SYE4 FMN. (a) Schematic representation of the chemical reduction of oxidized FMN (FMNO) to FMNH2 using NaBH4. For FMN, ‘R’ is ribityl phosphate. (B) Conformational plasticity of SYE4 FMN. The fully oxidized isoalloxazine ring is slightly bent in the apo form but planar when bound to TNP, likely as a consequence of π-stacking interactions. Upon two-electron reduction, the isoalloxazine ring system shows a more pronounced ‘butterfly bend’ around the FMN N5–N10 axis. Hydride (H:) transfer from the isoalloxazine ring to the electron-deficient aromatic nucleus of TNP and subsequent proton (H+) uptake produces the hydride-Meisenheimer complex (H-TNP). The regained planarity of the isoalloxazine ring indicates that hydride transfer is completed. 2mFo-DFc density, contoured at 1.0 σ for each structure, is shown as a blue mesh. mFo-DFc density, contoured at + 3.0 σ or − 3.0 σ, is shown as a green or red mesh, respectively. Positive and negative density peaks near the flavin cofactor might indicate a certain degree of X-ray-induced radiation damage. Electron density maps were calculated with the PHENIX suite [21]. Values for the isoalloxazine ‘butterfly bending’ are annotated next to each structure. ‘Butterfly bending’ is quantified by defining an angle between two planes fitted to either side of the ring system. These planes are defined by (A) atoms N10, N5, C2, and N3, and (B) atoms N10, N5, C7, and C8, respectively.

SYE4 in complex with trinitrophenol and crystallographic observation of its hydride-Meisenheimer complex

The key reaction step in the degradation of the TNT-analog picric acid (TNP) is the enzymatic hydride (H:) transfer to the aromatic nucleus (forming a hydride-Meisenheimer complex), followed by protonation. This step is then followed by enzyme-dependent elimination of nitrite [33]. Formation of a long-lived hydride-Meisenheimer complex (Fig. 4A) has been confirmed for three OYE homologs, PETN reductase (PETNR), xenobiotic reductase B, and N-ethylmaleimide reductase, which all form TNT hydride addition products. However, structural characterization of such complexes has remained elusive [34].

Details are in the caption following the image
Structural snapshot of a TNP hydride-Meisenheimer complex. (A) Schematic representation of the formation of the hydride-Meisenheimer complex of TNP. (B) Cutaway view of the SYE4 active site tunnel in the crystal structure of reduced SYE4 soaked with TNP. The electron density showed clear evidence for two TNP molecules; one stacked on the FMN, and one at the entrance of the tunnel. (C) Structure of TNP stacked on top of FMN in crystals of oxidized SYE4. (D) Structure of the hydride-Meisenheimer complex of trinitrophenol (H-TNP) after hydride (H) transfer from the FMN isoalloxazine ring to the electron-deficient aromatic nucleus of TNP in crystals of reduced SYE4. H-TNP shows a discretely disordered stacking on top of the FMN isoalloxazine ring; the separate conformers are colored yellow and salmon pink, respectively. The second TNP molecule forms a hydrogen bond with the amide nitrogen of Ala41 of a symmetry-related SYE4 molecule using its hydroxyl head group, and is also engaged in charged interactions with H-TNP. The second TNP molecule is also π-stacked with Phe273, revealing a probable mechanism of entry and/or release of nitro-aromatic substrates. (E) Detailed comparison of the stacking modes of TNP and H-TNP shows canonical stacking only in the case of H-TNP. For all figures, 2mFo-DFc density, contoured at 0.5 σ, is shown as a blue mesh, and mFo-DFc density, contoured at + 3.0 σ or −3.0 σ, is shown as a green or red mesh, respectively. Electron density maps were calculated with the PHENIX suite [21].

To visualize the formation of an initial hydride-Meisenheimer complex in the reductive detoxification of nitro-aromatic compounds by SYE4, we revisited a prior observation that SYE4 undergoes a drastic color change to dark orange upon long incubations with picric acid, which suggested stabilization of a hydride-Meisenheimer complex, albeit without concomitant enzymatic turnover to assign actual enzymatic activity of SYE against picric acid [11]. To this end, we incubated oxidized and NaBH4-reduced SYE4 crystals with TNP. In the resulting crystal structure, two TNP molecules could be identified; one stacked on top of the FMN, and one at the gorge of the active site tunnel (Fig. 4B). The conformation of the active site flavin was used to confirm the reduction reaction: the bent, NaBH4-reduced FMN (FMNH2) had completely regained planarity, confirming hydride transfer from FMNH2 to the aromatic nucleus of TNP (Fig. 3B). To our knowledge, this is the first structural documentation a Meisenheimer complex in complex with a flavoenzyme, and for that matter any enzyme carrying a cofactor. Furthermore, our structural analysis of SYE4 is together with two studies on glutathione S-transferase (PDB entries 1AQX [35] and 5IA9) only the second structural example of any Meisenheimer complex bound to any protein.

Interestingly, the binding mode of H-TNP differs from that of TNP bound to oxidized SYE4. In the oxidized state of SYE4, TNP interacts with the catalytic His/Asn pair using one of its nitro groups. In this binding mode, TNP is not perfectly stacked with the pyrazine moiety of the isoalloxazine ring (Fig. 4C,E). In contrast, H-TNP is hydrogen-bonded to the catalytic His/Asn pair via its hydroxyl group; this results in a stacking mode that resembles that of the phenolic ligands, with atom N5 of the pyrazine ring poised for hydride transfer to the TNP aromatic nucleus (Fig. 4D,E).

Ligand-unbiased difference maps showed the presence of a large peak at the exterior of the ring system of the H-TNP molecule in the active site. This likely represents the hydroxyl group of a second, minor TNP conformation and consequently, we modeled H-TNP as discretely disordered (Fig. 4D). Given the outward facing orientation of the hydroxyl group, this minor conformation might capture H-TNP during exit of the active site tunnel.

The second TNP molecule, located at the mouth of the active site, is engaged in charged interactions with the flavin-bound H-TNP. Also, it interacts with Phe273 via π-stacking and is hydrogen-bonded to the main chain amide nitrogen of Ala41 of a symmetry-related SYE4 molecule in the crystal. As the Phe273 side chain has flipped ~ 90° about the CαCβ axis compared to its conformation in the native, unliganded structure, Phe273 likely serves as a platform for guiding the entry and/or release of aromatic substrates (Fig. 4C,D). Similarly, Phe241, part of the 12-residue long loop linking β5 and α5, stabilizes the binding of TNP on top of the FMN by sandwiching it via π-stacking interactions (Fig. 4C,D).

Conclusion

The physiological importance of the OYE family remains enigmatic, but most probably lies in a general detoxification role [8, 36, 37]. The fact that a single organism encodes four distinct OYE enzymes is remarkable in its own right. Therefore, the presence of four OYE homologs in S. oneidensis tempts us to relate such apparent enzymatic redundancy to the high respiratory versatility of the bacterium. Indeed, use of such a large range of electron donors and acceptors [38] likely leads to high rates of production of radicals that must be scavenged or detoxified to prevent cellular damage. Of the four SYEs, SYE4 has the broadest substrate profile as exemplified by its activity toward nitroglycerine and ability to form complexes with nitro-aromatic explosives. Moreover, it is the only SYE induced under oxidative stress conditions [11]. In this paper, we visualized how the active site architecture of SYE4 is poised for binding diverse ligands and we also identified a likely route for the entry of long-chain hydrophobic substrates. We have also been able to structurally characterize at high resolution the fortuitous stabilization of a Meisenheimer complex of picric acid in the active site of SYE4, albeit as a catalytically stalled state, thereby providing for the first time how the structural and electronic plasticity of a flavin cofactor provides the necessary platform to bind such a complex chemical adduct in an enzyme active site. We envisage that such information may provide insights into how flavin cofactors and engineered enzymes can be exploited toward chemical bioremediation approaches. Taken together, our work highlights the chemical promiscuity of SYE4 and supports a general detoxification function for SYE4 in S. oneidensis.

Accession numbers

Coordinates and structure factors have been deposited in the PDB (www.rcsb.org) with accession numbers 4B5N, 5K1K, 5K1M, 5K1Q, 5K1W, 5K0R, and 5K1U.

Acknowledgements

We thank the SWISS LIGHT SOURCE (SLS) and DESY-EMBL for synchrotron beam time allocation, and the staff of beamlines X06SA (SLS) and X11 and X13 (DESY-EMBL) for technical support. Access to these synchrotron facilities was supported by the European Commission under the 7th Framework Programme: Research Infrastructures, Grant Agreement Number 226716. This research project was supported by grants from the Research Foundation Flanders (FWO) and Ghent University (BOF instrument) to SNS. JE was supported by a research fellowship from the FWO.

    Author contributions

    JE, AB, JVB, and SNS designed research. JE, AB, and SNS performed experiments. JE, AB, and SNS analyzed data. JE and SNS wrote the manuscript.