The Crystal Structure of a Bacterial l-Arabinonate Dehydratase

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Crystal structure of a bacterial L-arabinonate dehydratase contains [2Fe-2S] cluster Mohammad Mubinur Rahman, Martina Blomster Andberg, Senthil Kumar Thangaraj, Tarja Parkkinen, Merja Penttilä, Janne Jänis, Anu Koivula, Juha Rouvinen, and Nina Hakulinen ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2017 Downloaded from http://pubs.acs.org on June 2, 2017

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ACS Chemical Biology

The first crystal structure of IlVD/EDD family member that contains a catalytically important [2Fe-2S] cluster and a Mg ion. 39x27mm (300 x 300 DPI)

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Crystal structure of a bacterial L-arabinonate dehydratase contains a [2Fe-2S] cluster

Mohammad Mubinur Rahman1, Martina Andberg2, Senthil Kumar Thangaraj1, Tarja Parkkinen1, Merja Penttilä2, Janne Jänis1, Anu Koivula2, Juha Rouvinen1 and Nina Hakulinen1*

1

Department of Chemistry, University of Eastern Finland, PO Box 111, FIN-80101 Joensuu, Finland

2

VTT Technical Research Centre of Finland Ltd, PO Box 1000, FIN-02044 VTT, Espoo, Finland

Keywords: L-arabinonate dehydratase, hydrolyase, iron-sulfur cluster, [2Fe-2S], pentose sugar acid, IlvD/EDD family

The authors declare no conflict of interest * To whom correspondence should be addressed: Nina Hakulinen, Department of Chemistry, University of Eastern Finland, PO Box 111, FIN-80101 Joensuu, Finland; Email [email protected]

Abstract We present a novel crystal structure of the IlvD/EDD family enzyme, L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT, EC 4.2.1.25), which catalyzes the conversion of L-arabinonate to 2-dehydro-3-deoxy-L-arabinonate. The enzyme is a tetramer consisting of a dimer of dimers, where each monomer is composed of


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two domains. The active site contains a catalytically important [2Fe-2S] cluster and Mg2+ ion, and is buried between two domains, and also at the dimer interface. The active site Lys129 was found to be carbamylated. Ser480 and Thr482 were shown to be essential residues for catalysis, and the S480A mutant structure showed an unexpected open conformation in which the active site was more accessible for the substrate. This structure showed the partial binding of L-arabinonate, which allowed us to suggest that the alkoxide ion form of the Ser480 side chain functions as a base and the [2Fe-2S] cluster functions as a Lewis acid in the elimination reaction.

\body Lignocellulosic biomasses provide a renewable source of sugars for future bio-refineries and contribute to the reduction of CO2 emission. Bioconversion of lignocelluloses through existing or synthetic microbial metabolic pathways allows production of biofuels and various platform chemicals to manufacture higher-value organic compounds or polymeric materials.1 The main focus, so far, has been in the utilization of cellulose (i.e. D-glucose) as a raw material, but pectin and hemicelluloses can also provide useful raw material for biorefineries. The aldopentoses, D-xylose and L-arabinose, are the most common building blocks in various hemicelluloses. Utilization of these pentose sugars by microorganisms can happen through various pathways.2 Some archaea and bacteria metabolize pentose sugars via a non-phosphorylative oxidative Dahms pathway, where L-arabinose is converted to pyruvate and glycolaldehyde in four steps.3 Alternatively, the oxidative pathway can lead to production of α-ketoglutarate via the so-called Weimberg pathway.4 An enzyme called L-arabinonate dehydratase (EC 4.2.1.25) (aka L-arabonate dehydratase or L-arabinonate hydrolyase) catalyzes in both of these pathways the conversion of L-


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arabinonate to 2-dehydro-3-deoxy-L-arabinonate (aka 3-deoxy-2-keto-L-arabinonate or 2keto-3-deoxy-L-arabonate) (Figure 1).

We have recently characterized from Rhizobium leguminosarum bv. trifolii the Larabinose/D-galactose 1-dehydrogenase (EC 1.1.1.85) that catalyzes the oxidation of Larabinose by NAD(P)H,5 as well as the L-arabinonate dehydratase (RlArDHT).6 These enzymes could be used, e.g., to produce 1,4-butanediol or glycolaldehyde that can be further converted to either ethylene glycol or glycolic acid.7,8 The RlArDHT was shown to dehydrate L-arabinonate, D-fuconate and D-galactonate in the presence of Mg2+. The enzyme was also shown to contain an iron-sulfur [Fe-S] cluster, and mutagenesis study further demonstrated that three conserved cysteine residues, Cys59, Cys127 and Cys200 in RlArDHT are needed for coordination of the [Fe-S] cluster.6

L-arabinonate dehydratases, similarly to other pentonate dehydratases, usually belong to the IlvD/EDD protein family, which includes various dehydratases involved either in amino acid or carbohydrate metabolism. IlvD refers to dehydratases in a branched-chain amino acid biosynthetic pathway and EDD refers to dehydratases in an Entner-Doudoroff pathway, which substitutes the classic glycolysis in some bacteria. Enzymes belonging to the IlvD/EDD family have been reported to contain either a [2Fe-2S] or a [4Fe-4S] cluster within the protein structure.6 Based on our recent phylogenetic analysis, the IlvD/EDD enzymes can be divided into at least six groups, where aldonic acid dehydratases form one group.6 Aldonic acid dehydratases of the IlvD/EDD family are rather unexplored and only a few of them have been characterized, i.e. the L-arabinonate dehydratase from Azospirillum brasiliense,9 D-gluconate dehydratase from Achromobacter xylosoxidans,10 and E. coli Dxylonate dehydratase,11 in addition to our recently characterized L-arabinonate dehydratase


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from R. leguminosarum bv. trifolii and a D-xylonate dehydratase from Caulobacter crescentus.6 Most of the known hexose sugar acid dehydratases such as D-gluconate and Dmannonate dehydratases belong to the enolase superfamily,12 which has been significantly more explored than the IlvD/EDD family. At present, the only available crystal structure of an enzyme that belongs to the IlvD/EDD family is the [Fe-S] cluster-free form of 6phosphogluconate dehydratase from Shewanella oneidensis (PDB code 2GP4). The crystal structure has been solved at 2.5 Å resolution within the Structural Genomics project, but no publication exists yet. 2GP4 model lacks the [Fe-S] cluster and some loops around the active site.

Here, we report the first crystal structure of a holo-enzyme, an L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT_holo), belonging to the IlvD/EDD family. In addition, an apo-structure without an [Fe-S] cluster (RlArDHT_apo) and the structure of an inactive S480A mutant (RlArDHT_S80A) were solved. The structure solving by molecular replacement was successful after the correction of 2GP4 model. The amino acid sequence identity of RlArDHT with 2GP4 is only 26% (SI Appendix, Figure S1). RlArDHT was found to form a homotetramer with a molecular mass of 260 kDa. The crystal structure showed that the enzyme has a catalytically active [2Fe-2S] cluster in its active site, as well as a Mg2+ ion, also needed for the activity. The structures allowed us to suggest a reaction mechanism for the enzyme.


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RESULTS AND DISCUSSION Overall structure In the RlArDHT crystal structures, the electron densities for amino acid residues 5-579 were seen explicitly, but the flexible N-terminus together with the fused Strep-tag was not visible in the electron density map. RlArDHT had a complex fold with no clear resemblance with any other determined protein structure except with the partially incorrect structure of the 6-phosphogluconate dehydratase from S. oneidensis (PDB code 2GP4). The analysis of RlArDHT by DALI server13 demonstrated significant structural similarity merely with this 2GP4 structure (Z-score 33, rmsd 2.9 Å). The next highest match (of the N-terminal domain) was found with phosphoribosylaminoimidazole carboxylase and N5carboxyaminoimidatzole ribonucleotide mutase (Z-scores 9-10) structures. Topology diagram of RlArDHT is shown in Figure 2c. The structure of RlArDHT can be considered to consist of two major domains: an N-terminal domain (residues 1-380) and a C-terminal domain (381-579) (Figure 2a). The active site is located in the deep pocket partially at the interface between two domains and also at the interface between the two monomers. The [2Fe-2S] cluster is bound to the N-terminal domain and the Mg2+ ion is located at the interface between the N- and C-terminal domains.

The core of the N-terminal domain is composed of an α/β open sheet structure with a fourstranded parallel β-sheet surrounded by α-helices on both sides, this could also be called a 3-layer-(αβα)-sandwich (residues 45-153, 196-251). In addition to the core α/β open sheet domain, the N-terminal domain of RlArDHT has three additional insertions: 1) an Nterminal helical structure (residues 1-44), 2) an insertion after the fourth β-strand containing β-hairpin motif and two α-helices (residues 154-195), and 3) a bundle of several α−helices (residues 252-380). The C- terminal domain contains a central mixed eight-


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stranded β-sheet barrel (residues 382-522) and a very long C-terminal polypeptide (523579), which wrap around both of the domains of the monomer. The last five C-terminal residues are close to the active site in the neighboring monomer.

At present, the description of the IlvD/EDD superfamily is based on the partially incorrect 2GP4 model in many databases, such as SCOP. This superfamily is described as having a N-terminal domain-like structure with a mixed β-sheet. However, our re-refined 2GP4 model and RlArDHT structures clearly showed that the N-terminal domain is composed of a parallel β-sheet of four β-strands.

The quaternary structure of RlArDHT is a homotetramer, which is composed of a dimer of dimers (Figure 2b). The overall dimensions of the tetramer were approximately 120 x 100 x 60 Å. Monoclinic RlArDHT crystals contained two tetramers (eight molecules A-H) in the asymmetric unit. Crystallographic symmetry operators in hexagonal and cubic crystals with dimer and monomer in the asymmetric unit, respectively, generated the same tetramer. This is consistent with the tetrameric oligomerization state determined by dynamic light scattering and by native mass spectrometry (Figure 3c and SI Appendix, Figure S2). Both the N- and C-terminal domains are involved in the large dimeric interactions. It is worth noting that the dimerization occludes access to the substrate-binding pocket (Figure 2b), which is located at the interface of two molecules in the dimer. The largest protein-protein interaction area is between molecules A and B with an interface area of 4020 Å2 as determined with a PISA server.14 Two dimers within a tetramer interact with each other via α-helix layer of N-terminal domains. The tetramer has a buried area of 23330 Å2 and a total surface area of 63630 Å2.


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A novel type of [2Fe-2S] cluster The electron density map of RlArDHT_holo suggested that the enzyme has a planar [2Fe2S] cluster (Figure 3, SI appendix Figure S3). This was repeatedly observed in all our crystal structures of RlArDHT in the holo-form. The iron-sulfur cluster exists in the Nterminal domain, where Fe1 is tetrahedrally coordinated by two bridging sulfide ions and two cysteines (Cys59 and Cys127), but Fe2 is only three-coordinated by two bridging sulfides and one cysteine (Cys200). The lack of the fourth ligand position leaves a vacant coordination site for Fe2 (Figure 3a). This is a very uncommon arrangement for [2Fe-2S]. Usually both irons in the [2Fe-2S] cluster are ligated to the protein by two amino acid residues, either cysteine or histidine residues.15,16 To the best of our knowledge, this is the first crystal structure having this type of scaffold in the [2Fe-2S] coordination and the first crystal structure of any hydrolyase (EC 4.2.1) containing a [2Fe-2S] cluster. One of the well-characterized iron-sulfur containing enzymes, the citric acid cycle enzyme aconitase, is known to have a [4Fe-4S] cluster ligated only by three cysteine residues, each of which ligates one iron and the remaining fourth iron binds to the substrate.17,18

The RlArDHT_holo structure had eight molecules (two tetramers) in the asymmetric unit, thus giving a possibility to estimate the variability of iron binding geometry (SI Appendix, Table S1). In chain B, a water molecule was refined close to Fe2 with a distance of 2.2 Å (Figure 3b) indicating that a solvent could complement the coordination number of Fe2 to four. A Tyr26 residue of chain A (dimeric counterpart) is 3 Å away from the water observed in chain B. The water was not clearly observed in other chains. It is likely that the loosely bound water is undergoing sufficient motion and therefore does not show up at the observed resolution. This labile water can be also easily replaced by the substrate. In the


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case of aconitase, the water is bound to Fe4 with a distance of 2.2 Å together with substrate making Fe4 six-coordinate with octahedral coordination geometry. 17,18

An L-arabinonate dehydratase from A. brasiliense (AbArDHT; sequence identity 61 % with our RlArDHT) has been claimed to have a [4Fe-4S] cluster based on EPR analysis and spectrophotometric characterization.9 On the contrary, Flint et al. have reported that some dihydroxy-acid dehydratases from bacteria have [2Fe-2S] cluster based on EPR and Uvvisible spectroscopy and they also predicted that AbArDHT would actually contain [2Fe2S] cluster. They suggested that Cys56, Cys124 and Cys197 are the [2Fe-2S] cluster coordinating residues in AbArDHT.19 Our crystal structure of RlArDHT now confirms these findings and shows that the corresponding residues in RlArDHT are Cys59, Cys127 and Cys200 as seen in Figure 3a. The sequence alignment of RlArDHT together with other IlvD/EDD members shows that Cys127 and Cys200 are totally conserved, but Cys59 is conserved only among the subgroup of this family (Figure 3e and Figure S1). It is very likely that all these enzymes including RlArDHT, AbArDHT and many bacterial dihydroxy- acid dehydratases have a functional [2Fe-2S] cluster. The members of another subgroup including a phosphogluconate dehydratase from Z. mobilis (Zm_6PGDT) and dihydroxy-acid dehydratase from E. coli (Ec_DHAD) have been reported to have a [4Fe4S] cluster.20,21 Molecular oxygen (or reactive oxygen species) has been reported to result in degradation of the catalytically active [4Fe-4S] to an inactive [2Fe-2S] cluster in some hydrolyases.22 However, the spinach dihydroxy-acid dehydratase has been shown to have a stable catalytically active [2Fe-2S] cluster.23 In RlArDHT, the [Fe-S] cluster seemed to be rather stable towards oxygen and we have successfully produced the active enzyme at aerobic conditions.6 The specific activity of aerobically expressed and purified RlArDHT showed


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some batch-to-batch variation (between 11 to 16 µmol min-1mg-1), but it maintained well its activity at pH 7.5-8.0 suggesting that the purified enzyme was stable. The colorimetrically quantified amount of iron was normally about 1.5 per a protein monomer, but never above 2. The dissolved RlArDHT_holo crystals were found to be completely active (Figure S4a), as the purified RlArDHT and the enzyme recovered from crystals showed specific activities of 16 and 13 µmol min-1mg-1, respectively. This further suggested that the [2Fe-2S] cluster, which is present in the crystal of RlArDHT, is the catalytically active form. In addition, anaerobic and aerobic reconstitution experiments resulted in a less active enzyme than the enzyme without reconstitution. The specific activity dropped from 16 to 2 µmol min-1mg-1, when 10 or 3 equivalents of iron per RlArDHT were used (Figure S4b). The UV-visible spectrum after the reconstitution (Figure S4c) showed an extra absorption band at 410 nm most likely due to the aggregation24 that might results to the inactivation of the enzyme. This was observed after the PD10 column purification. Similarly, it is reported that activity was dropped during the reconstitution experiments of dihydroxy-acid dehydratase from Sulfolobus solfataricus which also belongs to a [2Fe-2S] cluster containing enzyme group.19,25

The UV-visible absorption spectrum of RlArDHT also suggests the presence of the [2Fe2S] cluster (Figure 3d). The spectrum shows peaks at 325 nm (9900 M-1cm-1), 385 nm (6000 M-1cm-1), 440 - 480 nm (5000-4500 M-1cm-1) and 580 nm (2100 M-1cm-1) arising from S->Fe(III) charge transfer bands. Human ferrochelatase with a [2Fe-2S]2+ cluster has been reported to have absorption bands at 330 nm (ε = 24 000 M-1cm-1), 460 nm (ε = 11 000 M-1cm-1) and 550 nm (ε = 9000 M-1cm-1).26 We note that the [2Fe-2S] cluster in RlArDHT is ligated only by three cysteines, therefore the spectrum is not directly comparable to many of the previously published spectra from proteins that have [2Fe-2S]


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clusters. However, the UV-vis spectrum of RlArDHT is nearly identical to that obtained from spinach dihydroxy-acid dehydratase.23

The high-resolution ESI FT-ICR mass spectrometric analysis of RlArDHT in acidic conditions showed an existence of a major apo form and a minor form with bound [2Fe-2S] cluster and Mg2+ ion in a monomer with no evidence for other cluster forms (SI Appendix, Figure S5). Native MS experiments at pH 8.0 showed a peak corresponding to a tetrameric protein complex, which is fully consistent with the crystal structure. When holo-protein was oxidized with H2O2, a small but measurable mass decrease of ~351 Da was observed (Figure 3c). This would suggest a loss of four Fe and four S atoms per a tetramer, i.e., one Fe and one S per a monomer. It is likely that the sample initially contained a mixture of apo- (no cluster) and holo-forms (with [2Fe-2S] cluster). If RlArDHT would contain a mixture of several cluster forms ([4Fe-4S], [3Fe-3S] and [2Fe-2S], a larger mass decrease would be expected. Therefore, our conclusion is that the active form of RlArDHT contained [2Fe-2S] cluster, which can be inactived by generating the apo-form.

Iron-sulfur clusters are best known for their role in electron transfer, but have also been reported to have a role in enzyme catalysis, radical generation, stabilization of protein structures, sulfur donation, or to act as sensors for Fe or oxygen.27,28 The role of the ironsulfur cluster in L-arabinonate dehydratase is merely in enzyme catalysis and the [2Fe-2S] cluster is most likely inherently a non-redox type and probably also more resistant to oxygen damage. The reason for oxygen resistance may be due to the fact that the |Fe-S] cluster site, as detected in the RlArDHT_holo structure, is not solvent-exposed but completely buried due to the dimerization (Figure 2b).


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N6-Carboxy-lysine and the coordination of Mg2+ ion During the last cycles of the crystal structure refinement of RlArDHT_holo, the epsilon amino group of the active site Lys 129 was found to be converted to N6-carboxy-lysine (named as Kcx) (Figure 4). The same was later repeatedly observed in all crystal structures. This carbamylation of lysine is a post-translational modification, which has been found to occur spontaneously without the involvement of any specific enzyme.29 Since the resolution of the crystal structures was not very high, it was not easy to detect the modification, but a clear positive Fo-Fc density was seen on the tip of the Lys129 residue that suggested this residue had been modified (SI appendix, Figure S6). Based on the very positively charged environment of the site, it was deduced that the lysine residue was carbamylated. In RlArDHT, the N6-carboxy-lysine has a role in acting as an acidic residue, bridging to the Mg2+ ion and therefore being important for the catalytic function of the enzyme.

The Mg2+ has been shown to be absolutely essential for the activity of RlArDHT.6 The crystal structure of of RlArDHT shows that Mg2+ coordinated by Glu91, Asp128, Glu453 and Kcx129 residues (Figure 4). In addition, waters were found bound to Mg2+ ion, but some differences were observed between the molecules in the asymmetric unit of the RlArDHT_holo crystal structure (SI Appendix, Table S2). A square pyramidal geometry, where Mg2+ was ligated by four acidic amino acid residues and one water, was observed in molecules A, B, C, D and G (Figure 4a). In contrast, an octahedral geometry in which Mg2+ was ligated by four residues and by two waters was observed in molecules E, F and H (Figure 4b). The Mg2+ ion presumably has a common octahedral coordination, but the lack of a strong sixth coordination ligand might leave this site free for the substrate to bind. In addition, Glu91 residue can have alternative conformations.


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Apo-form lacking the [Fe-S] cluster and Mg2+ ion We also solved the crystal structure of apo-form (RlArDHT_apo) without a [2Fe-2S] cluster. In this crystal structure, Cys59 and Cys127 were found to form a disulfide bridge (Figure 4c). When cysteine residues are no longer bridged to a [2Fe-2S] cluster, the free thiolate anions close together can be readily oxidized to disulfides. The Mg2+ ion was also absent in the apo-structure and accordingly the residues Glu91 and Asp128, had adopted different conformation compared to that observed in holo-form. Furthermore, the position of the [2Fe-2S] cluster was replaced by the side-chain of Trp30 in the apo-structure (Figure 4c).

RlArDHT_S480A mutant co-crystallized with L-arabinonate The Ser480Ala mutant (RlArDHT_S480A) was totally inactive suggesting a crucial role of Ser480 for catalysis. Despite several soaking experiments with L-arabinonate and Dfuconate into crystals of RlArDHT wild type and RlArDHT_S480A mutant, the substrates were not found to bind into the active site. When the inactive RlArDHT_S480A mutant was co-crystallized with calcium arabinonate, a partially bound substrate was finally observed in the pocket (SI Appendix, Figure S7). The electron density map did not allow us to unambiguously refine the position of L-arabinonate, therefore it was excluded from the final crystal structure. The crystal structure of RlArDHT_S480A showed large conformational changes close to the active site and the structure can be considered to represent an open form of the enzyme. We want to note that the S480A mutation is not responsible of the conformational change, since we have solved a crystal structure of the S480A mutant (uncomplexed) also in the closed form.


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In the open form structure, the [2Fe-2S] cluster was only partially occupied (0.6) and consequently the linking cysteine residues (Cys59, Cys127 and Cys200) were refined in two conformations (0.6/0.4). The alternative conformations of Cys59 and Cys127 formed the disulfide bridge as observed in the apo-form. Thus, this structure represents a mixture of apo- and holo-form and it is likely that the open-form loses its iron-sulfur cluster more easily.

The observed partial electron density in the active site helped us to dock L-arabinonate into to pocket in a way that the carboxylate group of the substrate was bound to Mg2+ with a short distance of 1.9 Å. Similar binding of substrate to magnesium has been observed in members of the enolase superfamily.30,31 One oxygen atom of the carboxylate was located almost at the same position as the water molecule in the RlArDHT_holo structure. In addition, a hydroxyl group at C2 is 3.5 Å away from the Mg2+ and a hydroxyl group at C3 is 2.3 Å away from Fe2. The oxygen atom of the hydroxyl group at C2 in the modelled structure seemed to be slightly farther compared to the second water-based ligand of Mg2+ in the RlArDHT_holo structure. However, the resultant coordination geometry of Mg2+ is nearly octahedral.

The N-terminus of the α-helix and the preceding loop 200-204 participates in the formation of the substrate-binding cavity. In addition, the Mg2+ ion and its coordinating residues, particularly Asp128 and Glu453, are part of the binding cleft. A loop containing residues Ser480 and Thr482 also seems to be important for the formation of the substrate-binding cavity. In addition, the side-chains of residues Tyr26, Trp30 and His579 of molecule B protrude into the active site of molecule A and vice versa (Figure 5a). Therefore, the dimerization has a crucial role in the formation of the binding cleft. The hydroxyl groups of


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sugar acid tail (at C4, C5) seem to have weaker contacts with the enzyme than the head of the skeleton hydroxyl or acidic groups (at C1-C3). It is possible that the substrate might even adopt a different conformation, where the sugar acid tail reaches the residues Tyr26, Trp30 and His579. The contacts in the head region are shorter and most likely essential for all dihydroxy-acid dehydratases.

Conformational selection in L-arabinonate binding We were able to determine the enzyme structure in an open (S480A) and a closed (apo, holo) form. The root mean-square deviation (RMSD) between the two structures was 0.44 Å. The largest conformational difference was in the helix-loop-helix (Gly166-Ser192), which had tilted about 7 Å as a complete fragment (Figure 5b). This region had high Bfactors and the electron density map for side-chains of the residues from Thr167 to Lys177 was rather obscure, but it was clearly continuous for the main-chain atoms. The openingclosing motion does not occur at the dimeric interface, so the dimerization is therefore not altered. The shift opens the narrow access to the active site that enables the substrate to approach (Figure 5d).

The observed open and closed structures of RlArDHT may suggest that the enzyme utilizes the so-called conformational selection model in which the closed form is the dominant conformation for this enzyme in solution. The closed conformation increases the lifetime of the [2Fe-2S] cluster and thus the lifetime for enzymatic activity. The open conformation is required for the substrate binding but it may also lead to the loss of the [2Fe-2S] cluster as we observed. The population of open forms might be increased at alkaline conditions close to the pH-optimum of the enzyme. Crystals of open form were harvested from the crystallization drop at pH 8-8.5, which is the pH-optimum for RlArDHT.


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Proposed reaction mechanism RlArDHT can accept both pentonate and hexonate sugar acids as substrates with the highest activity on D-fuconate, L-arabinonate and D-galactonate. RlArDHT is strictly stereospecific at C2 and C3 for the R and S configurations, respectively, and the enzyme also has a clear preference for the S configuration at C4.6 Our crystal structure of the inactive Ser480Ala mutant docked with L-arabinonate showed that the substrate is bound to both the Mg2+ ion and Fe2. When the docked model and the crystal structure of RlArDHT_holo are superimposed, it can be seen that Ser480 is orientated towards the proton of the substrate on C2 and the hydroxyl group of the substrate on C3 is towards the Fe2 (Figure 5a). Therefore, the enantiomeric D-arabinonate is not able to bind. This is also confirmed by the fact that the enzyme does not catalyze dehydration of any sugar acids with configuration S and R at C2 and C3 positions, respectively. The specificity at C4 is not evident from the crystal structure and more studies are required to clarify it. The end of the sugar acid chain may even rotate into different orientations in the pocket. The active site cleft is long enough to allow the enzyme to act on sugar acids with various lengths. The interactions of the carboxylate group at C1 and OH groups at C2 and C3 are evident. This is due to the strong interaction of carbonyl with the Mg2+ ion and the involvement the [2Fe2S] cluster.

We suggest that the dehydration reaction begins by the abstraction of a proton from C2 by the alkoxide form of the Ser480 side chain. An oxygen atom OG of Ser480 is hydrogen bonded to the main chain nitrogen atom of Thr482 (3.1 Å) and the oxygen atom OG1 of Thr482 (2.7 Å). Therefore, threonine residue can keep serine residue in the correct position to react and it may also increases the nucleophilicity of serine oxygen. Our mutagenesis


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studies showed that both the RlArDHT_S480A variant and the RlArDHT_T482V are inactive. In addition, the acidity of the α-hydrogen might be increased when the substrate is bound to Fe2.

The formed carbanion can be stabilized by the Mg2+, which may reduce the electronegativity of oxygen on C2 and simultaneously weakens the C-O bond at C3. Fe2, which exists in the cluster in the Fe3+ oxidation state, acts as a Lewis acid that accepts the electron pair from the leaving hydroxyl group on C3 (Figure 6). Fe3+ is a strong Lewis acid with a tendency to combine with strong bases such as OH-. Whether this elimination reaction is an E1cb type stepwise reaction or concerted E2 type reaction is not clear. The elimination occurs in an anti-manner, like observed in aspartase/fumarase superfamily such as in fumarases, aconitases and enolases, which are assumed to follow the concerted E2 reaction mechanism.32 When the double bond is formed between C2 and C3, this enol intermediate is then subsequently tautomerized into the more stable keto-form either in enzyme or in solution.

Some hydrolyases, such as aconitase, use a protein bound [Fe-S] cluster to perform the Lewis-assisted catalysis.27 Aconitase catalyzes isomerization of citrate to isocitrate via a cis-aconitate intermediate in the tricarboxylic acid cycle. The isomerization requires dehydratation and rehydratation steps. The active form of the enzyme contains a [4Fe-4S] cluster, but by losing one of its irons an inactive enzyme with a [3Fe-4S] cluster is formed.33 The unique Fe can coordinate to a citrate substrate via Cβ-carboxyl oxygen and the Cβ-hydroxyl group (both at C3) and Ser642 abstracts the proton on C2.34 Mössbauer and EPR data has suggested that the cluster acts as a Lewis acid that accepts an electron pair from the citrate.35 It is also suggested that His101 may protonate the hydroxyl


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group.33,36 Our crystal structure of RlArDHT clearly supports the suggestion that iron-sulfur clusters in hydrolyases function as a Lewis acid and can facilitate the loss of a hydroxyl group from the substrate. However, RlArDHT is a new type of hydrolase with a [2Fe-2S] cluster and Mg2+ in its active site.

METHODS Recombinant RlArDHT was aerobically expressed in E. coli BL21 (DE3) cells. The enzyme was purified by Strep-Tactin affinity column followed by gel filtration, and crystallized by hanging-drop vapor diffusion method. Crystallization conditions for RlArDHT_holo were as described previously37 and the crystals of RlArDHT_apo and RlArDHT_S480A were obtained as described in SI Appendix Methods. The RlArDHT structure was solved by molecular replacement method using a corrected model of 6phosphogluconate dehydratase from S. oneidensis (2GP4) as a template. We found that the available model of 2GP4 was partly erroneous, which led to the necessity to re-refine the original 2GP4 model. More detailed experimental protocols can be found in SI Appendix, Table S3 and Methods.

Supporting Information The supporting information is available free of charge on the ACS Publication website. Sequence alignment, size distribution diagram, electron density maps for FeS cluster site, for carbamylated lysine and for L-arabinonate, enzyme activity graphs, UV-vis and mass spectra, bond distances, data collection and refinement statistics, and description of methods (PDF)

Accession Codes


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The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 5J83, 5J84 and 5J85 for RlArDHT_apo, RlArDHT_holo and RlArDHT_S480A, respectively.

Acknowledgements We wish to thank O. Liehunen (VTT) and P. Inkinen (UEF) for their technical assistance. We acknowledge the European Synchrotron Radiation Facility and Diamond Light Source for provision of synchrotron radiation facilities. The work at UEF was conducted with financial support from the Academy of Finland (decisions 256937 and 263931). At VTT, the work was part of the Finnish Centre of Excellence in the White Biotechnology-Green Chemistry programme (Academy of Finland decision number 118573) and the IV4SP project (Academy of Finland decision number 272598). The research that led to these results also received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570).

Author contributions M.A. and M.M.R. expressed and purified the protein samples. M.M.R. and N.H. crystallized the protein, collected the crystallographic data and solved the crystal structures. The mutants were designed by M.A., A.K. and N.H. Mass spectrometry analysis was carried out by S.K.T. and J.J. All authors have contributed to the structure analysis and the preparation of the manuscript.

Competing financial interest The authors declare no competing financial interests.


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FIGURE LEGENDS

Figure 1. The non-phosphorylative metabolic pathways of L-arabinose. In the Dahms pathway (shown in black) the final products are pyruvate and glycolaldehyde. In the Weimberg pathway (shown in blue) the final product is α-ketoglutarate. The enzymatic reaction, which is focused upon in this study, is framed.

Figure 2. a) Monomeric unit of RlArDHT, which is composed of two domains. N- and Cterminal domains are shown in light purple blue and dark blue, respectively. The [2Fe-2S] cluster (orange-yellow) and Mg2+ ion (purple) are shown as spheres. b) Oligomerization of RlArDHT. RlArDHT monomer (in blue) packs against the second monomer (cyan) forming a strong dimer. This dimer packs against a similar second dimer (grey and pink) forming the final tetrameric structure. The figure was created with PyMoL38 c) Topology diagram of the RlArDHT. The diagram was created using TopDraw.39

Figure 3. The observed [2Fe-2S] cluster a) in molecule A and b) in molecule B of the RlArDHT_holo structure. Omit map for the [2Fe-2S] cluster is shown in blue, contoured at 3.0 σ level. c) 12-T ESI FT-ICR mass spectra of RlArDHT in 20 mM ammonium acetate buffer (pH 8.0) for the holo-protein (cyan) and the oxidized apo-protein (green), with their masses (averaged over the all detected charge states ± SD) indicated. Numbers (n+) indicate protein ion charge states. d) UV-Vis spectra (300–800 nm) for the holo-protein (cyan) and oxidized apo-protein (green). e) Multiple sequence alignment of cluster binding cysteine residues within IlvD/EDD family enzymes. [2Fe-2S] cluster binding residue Cys59 is only conserved among subgroup of IlVD/EDD family, while residues Cys127 and Cys200 are totally conserved. The diamond indicates the cluster coordinating residues, and


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the asteric shows the totally conserved residues. The graphical sequence alignment was prepared using ALINE.40

Figure 4. The coordination of the Mg2+ ion a) as seen in molecule A and b) as seen in molecule E. The epsilon amino group of the active site Lys 129 was found to be converted to a carbamate (named as Kcx) c). Superposition of the actives sites of RlArDHT_holo (shown in grey) and RlArDHT_apo (shown in green) structures.

Figure 5. a) The substrate-binding pocket. L-arabinonate is docked into the RlArDHT_holo structure. Hydrogen atoms of the substrate are also shown. Ser480 abstracts the proton from the C2 of L-arabinonate. b) Superimposition of a closed form and an open form of an RlArDHT

monomer.

The

closed

form

(RlArDHT_holo)

and

the

open

form

(RlArDHT_S480A) are shown in blue and grey, respectively. L-arabinonate at the catalytic site is also represented as a stick model. Major conformational changes are highlighted. c) Surface representation of the closed form. The dimeric counterpart is shown in cyan. No entry into the active site. d) Surface representation of the open form. There is a narrow but free access from the protein surface into the active site.

Figure 6. A proposed reaction mechanism for L-arabinonate dehydratase.


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Figure 1


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Figure 2 140x155mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3


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Figure 4



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Figure 5


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Figure 6 65x61mm (300 x 300 DPI)


The Crystal Structure of a Bacterial l-Arabinonate Dehydratase

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