A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds

Class II terpene cyclases, such as oxidosqualene and squalene-hopene cyclases, catalyze some of the most complex polycyclization reactions. They minimally exhibit a β,γ-didomain architecture that has been evolutionarily repurposed in a wide range of terpene-processing enzymes and likely resulted from a fusion of unidentified monodomain proteins. Although single domain class I terpene cyclases have already been identified, single domain class II terpene cyclases have not been previously reported. Here we report high-resolution X-ray structures of a monodomain class II cyclase, merosterolic acid synthase (MstE). With a minimalistic β-domain architecture, this cyanobacterial enzyme is able to construct four rings in cytotoxic meroterpenoids with a sterol-like topology. The structures with bound substrate, product, and inhibitor provide detailed snapshots of a cyclization mechanism largely governed by residues located in a noncanonical enzyme region. Our results complement the few known class II cyclase crystal structures, while also indicating that archaic monodomain cyclases might have already catalyzed complex reaction cascades.

Terpene cyclases are key biosynthetic determinants for the vast diversity of isoprenoids, currently encompassing more than 76,000 compounds (Dictionary of Natural Products. http://dnp.chemnetbase.com). These enzymes convert acyclic substrates to cyclized products that can feature multiple new stereocenters and carbon-carbon bonds 1,2 . Among the two major classes of terpene cyclases 3 , class I enzymes dissociate pyrophosphorylated substrates to generate a carbocation for cyclization 4 , while class II enzymes protonate olefinic or epoxide moieties 5 . In canonical type II cyclases, an Asp residue in a conserved DXDD motif is responsible for Brønsted acid catalysis 6 . Available cyclase structures contain up to three domains, termed α, β, and γ ( Fig. 1) [7][8][9] . Single-domain architectures are currently only known for the α-domain microbial class I terpene cyclases [10][11][12][13] . Other class I enzymes feature αβ or αβγ architectures, with the α-domain containing the active site [14][15][16] . For class II terpene cyclases, which generate some of the most complex terpene skeletons, only βγ or αβγ architectures are known [17][18][19][20] It has been proposed that plant αβγ diterpene cyclases evolved from bacterial progenitors by fusion of the α and βγ enzymes and subsequently gave rise to αβ-didomain synthases by loss of the γ domain 7 . The class II βγ enzymes likely evolved from monodomain proteins, but as the catalytic acid residue is located in the β-region, and the γ-region provides most of the remaining residues needed for polycyclic

Results and discussion
The structure of apo merosterolic acid synthase Here we report merosterolic acid synthase (MstE) as a founding member of the monodomain class II cyclases and present detailed structural and functional insights into the formation of its polycyclic steroid-like product. MstE constructs the core scaffold of the cyanobacterial cytotoxin merosterol A ( Fig. 2A, 1), isolated from Scytonema sp. PCC 10023, by generating the pentacyclic intermediate merosterolic acid A (2) 21 . Although MstE produces a steroidlike skeleton, it does not act on a squalene-derived substrate but diverts the ubiquinone-type precursor 5-geranylgeranyl-3,4-dihydroxybenzoate (GG-DHB, 3) to specialized metabolism. X-ray structure analysis of MstE at resolutions up to 1.3 Å revealed atomic insights into this complex reaction (Supplementary Table 1). The monomeric state of MstE was confirmed by PISA calculations and size-exclusion chromatography 22 . It shows sequence homology to triterpene cyclases, such as squalene-hopene cyclases (SHCs, PDB-ID: 1H3B 23 , 2SQC 24 ) and oxidosqualene synthases (OSCs, 1W6K 18 ), classifying MstE as a class II cyclase (Extended Data Fig. 1-2). It is, however, significantly smaller than the homologs ( Supplementary Fig. 1). MstE exhibits a conserved α 6 -α 6 barrel fold with outer (o) and inner (i) helices forming a tilted circular arrangement (base subdomain, Fig. 3A).
Interestingly, helix oH 1 of MstE corresponds closely to the N-terminal helix of the β-domain in canonical βγ-didomain triterpene cyclases (Extended Data Fig. 1). While the γ-domain of SHCs and OSCs mediates association and product incorporation into membranes, MstE solely uses the pronounced coils between each o and i helix (head subdomain, Fig. 3B) to form an extended central cavity about 17 Å deep and 6 Å wide (Fig. 3C). Residues 157 to 165, at the entrance of the head subdomain's proposed terpene-binding site, are disordered in the apo structure (1.95 Å, PDB ID: 6SBB). Being lined with aliphatic and aromatic residues, the channel displays a negative electrostatic potential attracting hydrophobic molecules from the crystallization conditions (e.g. PEG) in absence of a natural ligand. Between the base and head subdomains, two Q-X 5 -W motifs located at the C-termini of oH 4 and oH 6 generate additional stability to endure the exergonic reaction. Found commonly in SHCs and OSCs 17,25 , they consist of tryptophan residues stacking with glutamine sidechains that form H-bonds towards the neighboring helix.

Closed-state structure of MstE with non-reactive substrate
First insights into the reaction cascade were gained by the co-crystal structure of MstE and non-reactive pseudo-substrate farnesyl dihydroxybenzoate (FS-DHB, 8) at 1.35 Å (PDB ID: 6SBC, Extended Data Fig. 3B, Supplementary Fig. 2, 3). The surrogate is fully defined in the 2F O -F C electron density map and located inside the binding channel with its C15-isoprenylic moiety oriented towards the protein core. Both hydroxy groups in DHB form strong H-bonds with Glu339. Upon ligand binding, residues 157 to 165 fold around the entrance in a substrate-induced fit, preventing access of solvent that could quench carbocations during the reaction. This is predominantly mediated by interactions between the DHB carboxylate and Tyr157 as well as the salt bridge between Asp162 and Arg337. Based on the structure of MstE:8, single-point mutants with impaired activity were constructed. Based on the protein alignment Asp109 was the likely protonating residue and was therefore chosen for mutagenesis (Extended Data Fig. 1). Tyr157 was selected for its interaction with the carboxylic acid of the DHB and Glu339 for its H-bonds with the DHB hydroxyl groups. Arg337 was chosen because of its interaction with Glu339.

Structures of mutant MstE with natural substrate and product
Crystallization trials in presence of the natural substrate GG-DHB with diffuse electron density in the prenyl-binding cavity and supports the role of Tyr157 as a gatekeeper for substrate recognition (Extended Data Fig. 3F). 3 was identified in MstE_R337A (Extended Data Fig. 3E), but the lack of stabilization at the entrance resulted in low ligand occupancy. Intriguingly, MstE_D109N forms a complex with 3, where the ligand occupies the binding pocket entirely (Fig. 3D, Extended Data Fig. 3C, Supplementary  Fig. 2). In accordance with the stereoisomeric configuration of the polycyclic product, all isoprenyl segments are forced into chair-like conformations. Together with the one-sided parallel alignment of all methyl groups, this compressed shape provides significant catalytic energy. In the superposition of MstE_D109N:3 and MstE:8 (RMSD-C α : 0.12 Å) N109 takes the exact position of D109 and rests perpendicular to the terminal C=C bond of 3 at 3.2 Å. The residue is positioned in a DXD-polar motif conserved among class II cyclases and known to initiate olefin protonation 8 . Unexpectedly, MstE_D109A co-crystallized with 3 revealed cyclized product MA (2) in the binding pocket (Fig. 3E, Extended Data Fig. 3D, Supplementary Fig. 2). Presumably, the energy contribution through substrate coordination and product stabilization is sufficient for a minimal turnover within the time frame of crystallization experiments over several days ( Supplementary Fig. 4). While the linear substrate observed in MstE_D109N:3 occupies the entire channel with a length of 17 Å, 2 is condensed to 12 Å and bent by methyl repulsions. Notably, 2-methyl-2,4-pentanediol (MPD) from crystallization buffers occupies the vacant space and seals the channel bottom. This might aid the selective binding of product even in excess of substrate.
Simultaneously, the DHB moiety of 2 loses H-bond contact to Glu339 at the meta-hydroxy group (Fig. 3E), and the carboxylate leaves the aromatic plane due to steric clashes with the generated C5-ring. As a result, the gatekeeper Tyr157 turns by 90° and Arg337 is displaced, forming a salt bridge with Glu339. The collective effect disorders the region between residues 330 and 335, supporting product release.
The structural snapshots at atomic resolution suggest that Asp109 initiates the reaction with its proton oriented in anti-configuration. This activated Brønsted acid forms a charge-relay system with Tyr216 and two water molecules (Extended Data Fig. 4, Supplementary Fig.  5). After protonation of the terminal C=C bond in 3 and concomitant formation of the first cycle 26,27 , the acid is likely regenerated by bulk water through solvent-accessible channels. During the subsequent cascade, precisely positioned aromatic residues of the binding pocket would stabilize intermediate carbocations until final cyclization onto the DHB π-system 28 . Mutation of aromatic residues likely fulfilling this role in MstE were mutated, resulting in enzymes no longer converting substrate to any product ( Supplementary Fig. 6). Based on the MstE_R337A structure, the strong salt bridge between the Arg residue and Asp162 increases the basicity of Glu339, which deprotonates the DHB meta-hydroxy group, pushing π-electrons into the ring (Extended Data Fig. 5). Subsequently, spontaneous deprotonation at C6 and keto-enol tautomerism restores the aromatic system ( Fig. 2A).

Mutant MstE with expanded product specificity
Members of the triterpene synthase family generate a plethora of structural scaffolds based on active-site geometries, with particularly impressive diversification found in plants 29 . This raises prospects that our structural insights might facilitate engineering of soluble cyclases yielding modified products. Therefore, we screened structure-guided mutants of MstE for alterations to the polycyclization reaction ( Supplementary Fig. 6, 7). Among these, MstE_Y157A, predicted to exhibit weakened interactions with the DHB head group, formed a new product 9 after incubation with GG-DHB (Supplementary Table 2, Supplementary  Fig. 8-16). Characterization of 9, termed merosterolic acid B, by NMR identified a tricyclic isoprenoid moiety containing an exo-methylene group. The structure is consistent with the proposed role of Y157 and suggests that dislocation of the DHB π-system prevents attack by the third-ring carbocation intermediate, resulting in deprotonation as an alternative reaction path (Fig. 4). Interestingly, the artificial compound 9 resembles fascioquinols (10-11), meroterpene antibiotics reported from a deep-water sponge 30 .
SHCs are ancient enzymes that already existed 2.7 billion years ago and have been regarded as the origin of the βγ-didomain fold 31 . While the β-domain is preserved in most terpene cyclases (Fig. 1), MstE is active despite lacking a γ-domain, present in all other characterized di-and triterpene cyclases, revealing that class II synthases can assemble complex polycyclic scaffolds with monodomain architectures. It remains unclear whether the βγ-fold evolved through insertion of a γ-domain between oH 1 and iH 2 of an ancestral protein, or whether MstE derived from a βγ predecessor by the deletion of helices 2-13. To date, triterpene synthase-ligand interactions have only been reported for SHC bound to an inhibitor and OSC complexed with its product 17,18 . This study on MstE visualizes crucial steps in terpenoid biosynthesis at atomic resolution. The availability of a soluble cyclase that accepts pyrophosphate-free substrates offers augmented perspectives for chemoenzymatic synthesis using engineered enzymes as shown for MstE.

Recombinant protein expression and purification for crystallization
Transformed E. coli BL21 (DE3) strains (Novagen/Merck, Darmstadt, Germany) was grown in 2 L lysogeny broth (50 mg/L kanamycin) at 130 rpm and 37 °C until an OD 600 of 0.6 was reached. Expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and occurred over night at 20 °C. After harvest by centrifugation, cell pellets were washed in 0.9% (w/v) NaCl and stored at -20 °C. Cell pellets were resuspended in 60 mL buffer A (100 mM Tris/HCl, pH 7.5, 500 mM NaCl, 20 mM imidazole, 10 mM β-mercaptoethanol) with 1 mg DNAse I and 5 mg Pefabloc (Sigma-Aldrich, Taufkirchen, Germany) and disrupted by sonication with a Branson Digital Sonifier 250 (G. Heinemann, Schwäbisch Gmünd, Germany). After centrifugation (40.000 × g, 30 min, 4 °C) the supernatant was applied to a 5 mL HisTrap HP column equilibrated with buffer A using an ÄKTA prime plus system (GE Healthcare, Freiburg, Germany) at a flow-rate of 5 mL/min. The column was washed with 10 CV of buffer A prior to elution with a gradient of buffer B (0 -100% in 50 mL). After addition of 0.5 mg SUMO protease (Ulp1 from S. cerevisiae), the eluate was dialyzed against 5 L buffer C (20 mM Tris/HCl, pH 7.5, 100 mM NaCl, 1 mM βmercaptoethanol) overnight at 4 °C. Affinity chromatography was repeated, flow-through and wash fractions were collected and concentrated to 2 mL using Vivaspin ultrafiltration centrifugal concentrators (Sartorius, Göttingen, Germany). Protein concentrates were subjected to size-exclusion chromatography with a HiLoad Superdex 200 16/60 column equilibrated with buffer D (buffer C with 5 mM dithiothreitol instead of β-mercaptoethanol) on an ÄKTA purifier system (GE Healthcare, Freiburg, Germany) at 1.5 mL/min. Peak fractions were analyzed by SDS-PAGE, concentrated to at least 40 mg/mL using centrifugal filters and either applied directly in crystallography or flash frozen in liquid nitrogen and stored at -80 °C. Table 3 by the sitting drop vapor diffusion method. Co-crystallization was performed by adding 2 mM (final concentration) of the potential ligands. Crystals adequate for structure determination were soaked with cryo-protectant (50% reservoir solution with 50% glycerol) and frozen in liquid nitrogen. Diffraction datasets at 1.0 Å were obtained at the X-ray beamline X06SA (Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland). Diffraction intensities were evaluated by the XDS software package 32 . Molecular replacement was performed with Phaser by Patterson function calculations using coordinates of a squalene-hopene cyclase (PDB ID: 2SQC) truncated to a poly-alanine chain 24,33 . Restrained refinement of the structure with Refmac5 was alternated by model building with Coot 34,35 . ARP/ wARP solvent was applied to add water molecules prior to finalization by TLS (Translation/ Liberation/Screw) and restrained refinements until adequate values for R work and R free as well as all geometric parameters were reached (analyzed by the MolProbity webserver) 36

Cloning, expression and purification of MstE for mutant screening
To generate point mutants of MstE, pET-29b MstE was used from the previous study 21 . Point mutations were introduced with the QuikChange mutagenesis method (Agilent Technologies) and confirmed by sequencing. Primers were designed using the PrimerX tool (http://www.bioinformatics.org/primerx/). The generated plasmids containing mutant MstE were transformed into E. coli BL21 tuner (DE3) cells (Novagen). For expressions, cells were grown in terrific broth supplemented with 50 μg/mL kanamycin to an OD 600 of approximately 0.8 at 30 °C, induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 16 °C for another 20 h. Cells were harvested by centrifugation (20 min, 5000 × g, 4 °C), resuspended in buffer (50 mM phosphate buffer pH 7.8, 300 mM NaCl, 10 mM imidazole) and lysed by sonication (10 × 10 s pulses with 10 s breaks, on ice). After centrifugation at 15000 × g and 4 °C for 30 min, MstE was purified from the supernatant with Ni-NTA (Macherey-Nagel). The column was washed with buffer containing 20 mM imidazole and the protein was eluted stepwise with buffer containing 100 mM imidazole followed by 200 mM imidazole. Elution fractions were run on an SDS-PAGE and used for enzymatic assays directly.

Enzymatic assays
For quick assays, 1 mM of 3-geranylgeranyl-4,5-dihydroxybenzoic acid (3, 100 mM stock in DMSO, synthesized in previous study 21 ) was dissolved in 50 μL buffer (50 mM phosphate pH 7.8, 300 mM NaCl). 50 μL of the protein elution fraction was added and the mixture was incubated at room temperature for 16 h.
The assays were quenched with 1 μL formic acid (FA) and extracted 3 times with 100 uL ethyl acetate. Organic phases were combined, dried in vacuo, resuspended in 100 uL methanol and analysed by liquid chromatography/high-resolution mass spectrometry (LC/ HRMS) on a Thermo Scientific Q Exactive mass spectrometer coupled to a Dionex Ultimate 3000 UPLC system. We used a Phenomenex Kinetex C18 (4.6x150 mm) with a gradient from 5% acetonitrile + 0.1% FA in water + 0.1% FA to 100% acetonitrile + 0.1% FA in 10 minutes. To minimize substrate inhibition, protein concentrations were set to 200 μM and substrate concentrations to 0.5 mM.

Synthesis General Procedures
For the synthesis of 8 the procedure described by Lang M. and Steglich W. (2005) was followed 42 . For detailed procedures see supplementary information.

Purification of merosterolic acid B and NMR analysis
The ethyl acetate extracts of large-scale assays were separated by reversed-phase HPLC (Phenomenex Kinetex C18, 10 × 250 mm) with an isocratic run of 85% acetonitrile + 0.1% formic acid in water + 0.1% formic acid and a flow rate of 2 mL/min. The fraction containing the compound of interest was further purified by reversed-phase HPLC (Phenomenex phenyl-hexyl, 10 × 250 mm) with an isocratic run of 73% acetonitrile + 0.1% formic acid in water + 0.1% formic acid. Purifications from assays containing approximately 700 mg of MstE Y157A yielded 300 -400 μg of product. NMR spectra were recorded on a Bruker Avance III spectrometer equipped with a cold probe at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR. For details on the structure elucidation see supplementary information.

Protein alignments and phylogenetic analysis
Protein sequences were retrieved from the UniProt database, and β domains were identified based on alignment to available crystal structures for each domain architecture (MstE from this study, 1SQC, 1W6K, 2ONG, or 3PFP). Sequences corresponding to the β domain and up to 10 residues on either side were aligned using MUSCLE 43 . MEGA 7 was used to generate the tree (Extended Data Fig. 2) from the alignment using the Maximum Likelihood method based on the Whelan and Goldman model 44,45 . All positions with less than 20% site coverage were eliminated, and the resulting tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 3.5036)). Maximum-likelihood tree of β domains from functionally characterized and unknown terpene cyclases, as described in the Materials and Methods above. Organisms are colored according to the phylogeny of their origin: Protists grey, animals gold, fungi red, plants green, and bacteria blue. Terpene enzyme class, domain architecture, and product are also indicated.  The structure of MstE_D109N:GG-DHB reveals the formation of a triad between Glu339, Arg337 and Asp162. Asp162 forms a salt bridge with the guanidino group of Arg337. This pushes electrons towards the secondary amine which in turn polarizes the carboxylate of Glu339, strengthening the H-bond with the meta-hydroxy group of DHB and increasing basicity. After deprotonation, this cluster acts as a proton-relay, before it is promptly broken and reordered to accommodate the product. Heteroatoms are highlighted in red (O) and blue (N); the proton (light blue) was added with PyMol. Interactions are represented as dashed lines (distance given in Å).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.