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Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase – Nature Chemistry

SOI is a trimer with a ferric haem b prosthetic group

SOI is the most active enzyme and the only membrane-bound enzyme in the styrene side chain degradation pathway (Extended Data Fig. 1b), and it has become a promising candidate protein for large-scale applications of Meinwald rearrangements in industrial biocatalysis. To identify an enzyme homologue suitable for structural studies, we screened SOI proteins from several bacterial species for their recombinant protein expression levels as well as their solubility and stability in different detergents (Extended Data Fig. 2). These experiments revealed Pseudomonas sp. VLB120 SOI (UniProt ID O50216) as the candidate enzyme with the highest protein yields and the best detergent-solubility and detergent-stability properties (Extended Data Fig. 3a–c).

In the literature, SOI is described as a cofactor-independent enzyme9,19; however, our studies revealed an unexpected red colour (Extended Data Fig. 3a) associated with SOI expression and purification, immediately suggesting the presence of iron. Chemical analysis of purified SOI by inductively coupled plasma optical emission spectroscopy (ICP-OES) confirmed that iron is bound to the recombinant enzyme in a 1:1 molar ratio. Consistent with these results, UV–vis spectra showed a peak maximum at ~421 nm. The spectrum shifted from 421 nm to 419 nm upon mixing the sample with the reducing agent sodium dithionite and an additional peak emerged at 558 nm, indicating the presence of a reducible haem b prosthetic group tightly bound to SOI (Extended Data Fig. 3d). We therefore provide experimental evidence that ferric haem b is a prosthetic group of SOI.

Circular dichroism (CD) spectroscopy was used to assess the folding and thermal stability of SOI. The far-ultraviolet CD spectrum recorded from the recombinant enzyme showed a substantial amount of α-helical structure at 25 °C, with characteristic minima near 208 and 220 nm. A mean molar residue ellipticity Θ [222] value of ~24,000 deg cm2 dmol−1 indicates a degree of α-helicity of ~60–70% for the protein. A ratio of >1 for the CD signals at 222 nm/208 nm might be indicative of supercoiling of the enzyme’s transmembrane (TM) α-helices, a feature that is characteristic of coiled coils20 (Extended Data Fig. 4a). The temperature-induced CD unfolding profile recorded from SOI at 222 nm exhibited a sigmoid shape typical of a two-state transition with a melting temperature of ~55 °C (Extended Data Fig. 4b).

The oligomeric state of the purified SOI was assessed by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS). For the recombinant enzyme, a molecular weight of 140 kDa was obtained, a value that suggests the presence of an SOI trimer (calculated molecular weight of 59 kDa) plus an n-dodecyl-βd-maltopyranoside (DDM) micelle (calculated molecular weight of 76 kDa) (Extended Data Fig. 4c). Oligomerization of SOI is also consistent with the presence of bands migrating at 17 and 32 kDa on SDS–PAGE gels (Extended Data Fig. 3b). Additional bands revealed by SDS–PAGE analysis that were approximately two and four times the size of the monomeric SOI have also been reported previously18.

Next, we indirectly measured the isomerization activity of wild-type SOI (SOI WT) and its mutant Y103A in the presence and absence of the inhibitor benzylamine by means of a coupled enzyme assay with excess phenylacetaldehyde dehydrogenase (EcALDH; Extended Data Fig. 5a,c). EcALDH was used because the Pseudomonas phenylacetaldehyde dehydrogenase is only poorly expressed and therefore results in poor yields. The kinetic parameters KM, Kcat and Kcat/KM of SOI WT and SOI in complex with the nanobody used for structure determination (see next section) are listed (Supplementary Table 1a). These experiments also revealed that benzylamine acts as a competitive inhibitor, increasing the apparent KM for styrene oxide in its presence while not having any effect on the maximum rate of reaction Vmax (Extended Data Fig. 5b).

Structures of SOI reveal a unique substrate binding pocket

Next, we aimed at structure elucidation of SOI by single-particle cryo-EM analysis. Because the small molecular weight of SOI of only 59 kDa is expected to represent a considerable challenge for a cryo-EM approach, we raised conformational nanobodies to increase its molecular mass. Conformational nanobodies were generated as described in ref. 21. We characterized all nanobody/SOI complexes using analytical SEC and activity assays. Of particular interest was a nanobody that, as judged by analytical SEC, mediated the formation of a bigger complex, possibly a SOI–nanobody (SOI–NB) hexamer, and led to a threefold higher catalytic efficiency (kcat/KM) compared to the wild-type enzyme (Supplementary Table 1a,b). The higher catalytic efficiency seems to be the result of a lower KM of the enzyme for styrene oxide caused by the nanobody. We reconstituted the SOI–NB complex with and without the competitive inhibitor benzylamine into nanodiscs (MSP1D1-filled Escherichia coli polar 169 lipids) and subjected the samples to extensive cryo-EM data collection and image processing. We obtained three-dimensional (3D) reconstructions of SOI–NB and SOI–NB–benzylamine (SOI–NB–BA) complexes at resolutions of 2.05 Å and 2.12 Å. A local resolution analysis of the cryo-EM density maps showed that the core transmembrane domain of SOI can reach a resolution of 1.5–1.6 Å (Extended Data Figs. 6 and 7, and Table 1) with resolved fine structural details including ordered water molecules (Extended Data Fig. 8). The two structures are almost identical. Accordingly, superimposition of SOI–NB and SOI–NB–BA over 1,572 Cα atoms yielded a root-mean-square deviation (r.m.s.d.) value of 0.044 Å.

The structures revealed an extended complex consisting of a dimer of two SOI–NB trimers in which the electron densities of SOI, the nanobody and the ferric haem b prosthetic group are well defined (Extended Data Fig. 8). Both structures have a length of 156 Å and a width of 15 Å (Fig. 1a). Formation of the dodecameric complex is mediated by three main interfaces. First, SOI trimer formation is directed by the ferric haem b prosthetic group that is located at the subunit interface. The location of haem b at the interface of subunits possibly explains the high thermal stability of SOI (Extended Data Fig. 4c). Second, all three variable regions CDR1, CDR2 and CDR3 of the nanobody interact with the periplasmic loops of two different SOI protomers (Fig. 2a,d). Amino-acid residues R28, F30, V31 and P33 of nanobody CDR1 (NB-CDR1, G25-A34) and R100, G101, S103, G104, E107 and Y108 of CDR3 (NB-CDR3, S99-Y108) interact with V32, G33, I42, E44, S50, P51 and E52 of periplasmic loop 1 (PL1, V32-E52) (Fig. 2b,c) of one SOI protomer (Fig. 2d). Amino acids T51, N53, W54, H55, H58 and S60 of nanobody CDR2 (NB-CDR2, T51-S60) form interactions with F108, S109, P110, R112, P118, N119, F121 and P123 of periplasmic loop 2 (PL2, F108-I126) (Fig. 2b,c) of a second SOI protomer (Fig. 2d). Third, two nanobodies interact with each other via their conserved C-terminal β-strand in an anti-parallel complementary manner engaging L16, V94, P114 and T116 (Fig. 2e).

Fig. 1: Cryo-EM structure of membrane-bound SOI containing a ferric haem b prosthetic group.
figure 1

a, Cryo-EM map and model of the SOI–NB complex structure at 2.05 Å resolution. Each subunit of the SOI trimer (blue, light blue, cyan) is bound to a nanobody (NB, orange or yellow) via the periplasmic loops. Densities for the MSP1D1 nanodisc and for the disordered regions of SOI (C terminus and N-terminal 6xHis-tag, are coloured in light grey. b, Views of the SOI trimer from the periplasm (top), in plane with the lipid bilayer (middle) and from the cytosol (bottom). The three ferric haem b molecules bound at the subunit interfaces of the SOI trimer are coloured in pink. c, Electron density features of the ferric haem b prosthetic group visualized as a surface map (upper panel, map contoured to σ = 15) and schematic representation of the ferric haem b prosthetic group.

Fig. 2: Structural organization of the SOI–NB complex.
figure 2

a, Side view of the SOI–NB complex. b, One nanobody binds to the periplasmic loops of two adjacent protomers. Periplasmic loop 1 (PL1) of one subunit is shown in grey and periplasmic loop 2 (PL2) of a neighbouring SOI protomer is shown in blue. c,d, Details of the interaction between the SOI and nanobody. c, Interacting amino-acid residues of PL1 and PL2 are shown in white and cyan, respectively. The nanobody is shown as a space-filling model in orange. d, Interacting amino acids of NB-CDR1, NB-CDR2 and NB-CDR3 are labelled in yellow, brown and orange, respectively. e, The interaction between nanobodies is mediated by four residues that are conserved among nanobodies.

The structure of the SOI trimer is reminiscent of a classical transmembrane channel with an ‘open’ conformation towards the periplasmic space and a ‘closed’ state towards the cytosol (Fig. 1a,b). The functional importance of this conformation is currently not known.

To assess whether the nanobody has an influence on the structure of SOI, we aimed to elucidate the enzyme structure alone. Despite numerous attempts, we were not able to obtain its structure in the absence of the nanobody. We therefore used an SOI model predicted by AlphaFold2 to identify potential conformational changes in the enzyme upon nanobody binding22. There is convincing evidence in the literature that AlphaFold2 can accurately predict haem proteins in the absence of the haem cofactor. For example, lack of the haem cofactor or its tetramerization partners, which are essential for folding, does not stop AlphaFold2 from perfectly predicting the fold of the haemoglobin α-chain23. AlphaFold2 learns structure prediction at the amino-acid residue contact level, without the need for folding information, and can therefore accurately predict a single-chain haemoglobin fold that would never exist on its own or in the absence of the haem cofactor in nature. Furthermore, haem proteins in general are accurately predicted by AlphaFold in the absence of the haem cofactor24,25. Based on these findings, we believe that the use of an AlphaFold2-generated model as a reference for the state without nanobody is justified. Overall, the per-residue confidence score (pLDDT) was >90, indicating the high quality of the predicted structure. Low pLDDT values were only obtained for the C terminus of SOI, indicating that it is mostly unstructured. The model indicated that there is sufficient space for a haem b molecule between the subunits of the enzyme. Cα superimposition of the AlphaFold2 model onto the cryo-EM structure of the SOI–NB complex resulted in an r.m.s.d. of less than 1 Å over 169 Cα atoms, indicating that there exists only one predominant conformation of this enzyme. This conclusion is strongly supported by the identical structures observed for SOI–NB and SOI–NB–BA complexes.

A DALI search with SOI did not result in any substantial structural similarities to other proteins, a feature that also reflects the uniqueness of the enzyme26.

SOI substrate binding mode supports its broad substrate scope

The cryo-EM structures revealed that 17 amino acids from five TM helices originating from two adjacent monomers form a 5.4-nm3-sized catalytic centre cavity containing the ferric haem b prosthetic group (Fig. 3a–f). Two separate hydrogen-bond networks around the cavity are seen in the structures. Network 1 is composed of 11 amino acids from one monomer (coloured in marine). Hydrogen bonds formed by amino acids N64 (TM2), D95, N99, Y103 and L104 (TM3) are shown in Fig. 3c. The hydroxyl group of Y103 also forms a 2.9-Å-long hydrogen bond (Fig. 3e) to the nitrogen atom of benzylamine, suggesting that network 1 plays an important role in substrate positioning. Consistent with this conclusion, mutation of amino-acid residues N64 and D95 to A substantially impaired the function of the enzyme, whereas substitution of N99 and Y103 by A resulted in complete inactivation of the SOI variants, demonstrating their importance in catalysis (Supplementary Table 1b).

Fig. 3: Substrate binding pocket of SOI.
figure 3

a,b, Side (a) and top (b) view of the ferric haem b binding pocket formed at the interface of two SOI subunits. c, The active centre Fe(III) is coordinated by an axial H58 at the fifth coordination site and the competitive inhibitor benzylamine at the sixth coordination site. d,e, Network of residues from two adjacent SOI subunits in apo (d) and benzylamine inhibitor (e) bound states. The electron densities for haem b and benzylamine are shown as surfaces. The electron densities of haem b and benzylamine are shown in a surface representation (map contoured at σ = 15). In the apo state, an additional unknown density bound to haem b (shown as a white surface) is observed, which might correspond to a water molecule that mediates an interaction of Fe(III) and Y103 in the apo state. f, A superimposition of the SOI–NB (apo) and SOI–NB–BA (competitive inhibitor) bound states of SOI shows no structural changes associated with binding a competitive substrate inhibitor. g, Ordered structural water molecules in the catalytic centre facilitate the interaction of SOI with haem b. h,i, Network 1 (cyan) is important for positioning the substrate (h) and network 2 (blue) is required for the orientation of H58 (i). Hydrogen bonds and distances of Fe(III) to coordination sites are indicated by dashed lines. Shown values are in Å.

Network 2 consists of eight amino acids from the adjacent monomer (coloured in yellow). The most prominent features of network 2 are two hydrogen bonds formed between the first nitrogen atom of the H58 side chain (ND1 of H58) and the main-chain carbonyl group of T20 and between the H58 main-chain carbonyl group and the OG (oxygen atom) of the T20 side chain. Both interactions appear to lock the conformation of H58 (Fig. 3f). The H58A variant could not be assessed because its mutation resulted in a colourless, inactive and presumably monomeric protein that was very prone to aggregation. This result suggests H58 as the key residue for coordinating the Fe (III) and that the presence of the ferric haem b prosthetic group is crucial for proper enzyme folding and stability. Thus, the ferric haem b prosthetic group represents a key structural and functional element of SOI.

Our structural findings suggest that the ferric haem b prosthetic group plays an essential role in substrate binding and catalysis. Fe(III) has six coordination sites that are occupied by four equatorial ligands in the porphyrin ring and the side chain of H58 of a neighbouring subunit oriented perpendicular to the porphyrin ring and anchoring the ferric haem b prosthetic group at the lateral trimer interface (Fig. 1b). The distance of Fe(III) to H58 is 2.2 Å (Fig. 3e). The sixth ligand site of Fe(III) serves as interaction site with suitable groups of substrate molecules. As seen in the SOI–NB–BA complex structure (Fig. 1a,b), the substrate binding site positions the aryl group plane of benzylamine above and parallel to the porphyrin ring plane. The nitrogen atom of benzylamine occupies the sixth coordination site of Fe(III) at a distance of 2.3 Å (Fig. 3e). The ferric haem b acts as a Lewis acid, and its interaction with the epoxide oxygen atom should be sufficient to promote epoxide ring-opening. The position of the iron ligand atom together with the orientation of the aryl group, determined by its contact with the porphyrin ring, constrains the torsion angle of the benzylic C–C bond of benzylamine and likewise for bound styrene oxide. The size and property of the binding site controls the size and chemical nature of possible epoxide substrates.

Next, we investigated the catalytic centre using EPR studies. The EPR spectrum of purified SOI WT is dominated by a low-spin (LS) signal with g values of gzyx = 2.97, 2.28, 1.45 that we designate as LS1 (Extended Data Fig. 9a). The LS1 signal is characteristic of a ferric haem protein with a bis-His or His-imidazole coordination27. It is therefore possible that LS1 represents an imidazole adduct of SOI that is still present after purification. SOI WT also exhibits high spin signals (small features between 1,000 and 2,000 G), which are attributed to 5-coordinate haem (Extended Data Fig. 9b). These results suggest that the iron ion of haem b of SOI WT is a mixture of 5-coordination and 6-coordination. This is consistence with our X-ray absorption near-edge structure (XANES) measurement of SOI WT at room temperature (Extended Data Fig. 10a,b). EPR, XANES and cryo-EM data therefore suggest that, in the absence of the substrate, the product or an inhibitor (see below), the 6-coordination site of iron can be occupied by small ligand.

The SOI Y103A mutant exhibits a different EPR spectrum compared to SOI WT (Extended Data Fig. 9b). The Y103A spectrum is dominated by a highly anisotropic LS (HALS) signal (gz = 3.32), which is absent in SOI WT, and only a minor LS1 signal remained.

The dominant HALS signal (gz = 3.32) for the benzylamine adduct that is seen with wild-type and mutant enzyme variants (Extended Data Fig. 9a) cannot be explained by the perpendicular imidazole ligand planes, but is consistent with previous observations of a HALS signal with a primary amine ligand28,29. We therefore attribute the HALS signal as the N-coordinated benzylamine adduct, which is consistent with the structure showing direct coordination of the haem iron by the ligand (Fig. 3c).

Addition of the substrate styrene oxide and product phenylacetaldehyde to SOI resulted in very similar spectra, which show the appearance of a new LS species (LS2, gzyx = 2.60, 2.17, 1.84) characteristic of an oxygen sixth ligand (Extended Data Fig. 9a), with a presumed His/O-substrate or product coordination. For the SOI Y103A variant, LS2 was not observed and only a very dominant LS1-like signal was observed at the expense of the HALS signal (not shown). The absence of LS2 in the Y103A variant is consistent with the role of Y103 to form a hydrogen bond with the coordinating oxygen atom of the substrate/product. This suggests that the substrate and product still bind the active site, but in a less productive fashion.

The oxidation state of the iron of the haem b prosthetic group is Fe(III), as determined by EPR (Extended Data Fig. 9). The Fe(III) state acting as a strong Lewis acid is crucial for SOI function. HS and LS signals originated from Fe(III) disappeared after reduction with excess sodium dithionite (Extended Data Fig. 9a). As a result, we do not know whether the substrate/product will still bind under reducing conditions.

Regio-selectivity and stereo-specificity of SOI

Docking experiments, guided by the benzylamine binding mode (Fig. 4a), with (S)- and (R)-styrene oxide (Fig. 4b,c), (S)-α-methylstyrene oxide (Fig. 4d) and (1S,2S)-trans-2-methyl-3-phenyloxirane (Fig. 4e) confirmed that the bound substrates are conformationally highly restricted, which is the basis of the high regio-selectivity and stereo-specificity of the subsequent reaction. The shown epoxides (and many more known to be good substrates) can be fit well in the substrate binding site with a distance of the epoxide oxygen to the iron atom around 2.3 Å and in hydrogen bond distance to the hydroxyl group of Y103. The binding mode is always similar to that of benzylamine (Fig. 4a) and is maintained with moderate positional adjustments when modelling a ring-opened sp2-hybridized carbocation intermediate.

Fig. 4: Docking experiments explain the substrate range and regio-selectivity and stereo-specificity of SOI.
figure 4

a, The substrate binding pocket occupied by benzylamine, as seen in the cryo-EM structure (left) and key interactions (right). be, Different substrates docked to the binding pocket. Left: (S)-styrene oxide (b), (R)-styrene oxide (c), (S)-α-methylstyrene oxide (SAMO) (d) and (1S,2S)-trans-2-methyl-3-phenyloxirane (MPO) (e). Right: key interactions.

Both (S)- and (R)-styrene oxide can be well fitted in positions essentially related by a mirror plane through their oxygen atoms and perpendicular to their overlapping phenyl rings (Fig. 5). The R enantiomer has been shown to react more quickly, but the difference is too small to be rationalized by static structural considerations. Similarly, the ring-opened sp2-carbocations of both enantiomers, assumed to be stabilized by co-planarity with the aryl ring and with their oxygens still ligated to the haem iron, can be modelled without causing substantial repulsive interactions and maintaining the approximate mirror relationship. Fixed in this relatively rigid conformation, only one of the two hydrogens of Cβ of the epoxide is in a favourable position to shift to Cα, explaining the high specificity of the reaction and the conserved chirality at Cα. At the same time, it explains why internal epoxides methylated at Cβ, such as (1R,2R)-2-methyl-3-phenyloxirane, gave only one diastereomer by shifting the methyl group in the trans-position, but not the other product by transferring the hydrogen in the cis-position11 (Supplementary Fig. 2a–f).

Fig. 5: Proposed SOI reaction mechanism for the isomerization of styrene oxide.
figure 5

a,b, Mechanisms for the isomerization of (S)-styrene oxide (a) and (R)-styrene oxide (b). Y103 positions the oxygen atom of the epoxide ring optimally for the Fe(III) of haem b to act as a Lewis acid, resulting in epoxide ring-opening, carbocation formation and a stereo-specific 1,2-hydride shift.

The mechanism of the Lewis acid-catalysed Meinwald rearrangement is commonly presented as being initiated by epoxide ring-opening (involving C–O bond-breaking and relaxation of the strained geometry), leading to a carbocation intermediate followed by a 1,2-hydride (alkyl) shift and product formation. Alignment and partial overlap of the occupied bonding orbital of the shifting nucleophile with the empty p-orbital of the carbocation is thought to be needed to permit the shift. However, as the carbocation is achiral, the stereo-specificity observed for the SOI-catalysed reaction would have to be due to an enzyme environment-mediated restriction of transfer to only one face of the carbocation plane. With α-methylstyrene oxide as substrate, one would thus expect that only one enantiomer is formed, independent of the chirality of the educt. Instead, both enantiomers react with retention of chirality, as reported by two groups15,16. Meza and colleagues16 therefore precluded the carbocation hypothesis and instead proposed a concerted Meinwald rearrangement where stereo-specificity is under substrate control. To us, a concerted C–O bond-breaking/hydride (or alkyl) shift appears stereochemically highly unfavourable (H–C–C–O torsion angle of ~110° rather than 180°) and is not compatible with the established antiperiplanar geometry of concerted bond-breaking/bond-forming rearrangements. As a 1–2 (equivalent to Cα–Cβ) bond rotation is not possible before the oxirane ring opens, an antiperiplanar orientation is not accessible. Furthermore, the very low stereo-specificity observed for the chemical Meinwald rearrangement of these substrates15 would imply that substrate control is only taking place in the enzyme environment. Our structural and modelling results reveal a different but very elegant solution to this problem that is fully consistent with the carbocation hypothesis. The key finding is that (R)- and (S)-styrene oxide bind in two different ways (related by an approximate local mirror plane) to the active site, as already described. As a result, the shifting group attacks from the same side with respect to the protein environment but from opposite sides if we take the prochiral carbocation as the reference frame. Further support for a carbocation intermediate is provided by the observation that aryl electron-donating and electron-withdrawing substituents in the para position lead to an accelerated and strongly reduced reaction rate, respectively30, consistent with their expected inductive effect on benzyl carbocation stability. With respect to the final electronic rearrangement, the conformation of our modelled carbocation intermediate shows a favourable, nearly antiperiplanar arrangement of the moving electron pairs (involved in hydride shift and C=O double-bond formation, respectively). We thus clearly favour the carbocation intermediate hypothesis as it fulfils established (stereo) chemical principles and is fully consistent with the observed enzyme stereo-specificity.