Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron(II) and 2-oxoglutarate-dependent dioxygenase EctD

Abstract

As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD) is a member of the non-heme iron(II)-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe(3+) at a resolution of 1.85 A. Like other non-heme iron(II) and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family.

Figure 1. Enzymatic reaction scheme for the EctD-mediated hydroxylation of ectoine.

Figure 2. Ribbon and surface representation of the EctD ectoine hydroxylase.

(A) The successive segments of the double-stranded β-helix (DSBH) are coloured according to the scheme of Branden and Tooze . The Fe³⁺ ion bound by EctD is shown as a blue sphere. A dashed line indicates a disordered loop region connecting the DSBH β-strands IV and V. (B) The surface of the EctD protein is represented and the side-chains of the iron-coordinating residues His-146, Asp-148 and Asp-248 are shown as sticks. The iron ion bound by the crystallized EctD protein is shown as a blue sphere.

Figure 3. Topology diagrams of PhyH-family enzymes PhyH, SyrB2 and EctD and of the DAOCS enzyme.

(A) PhyH (PDB code: 2A1X), (B) SyrB2 (PDB code: 2FCU) and (C) EctD (PDB code: 3EMR). (D) Topology diagram of DAOCS (PDB code: 1DCS), a representative of the family of the IPNS/DAOCS-like enzymes. Arrows represent β-strands, while α-helices are shown as cylinders. 3₁₀-helices are also shown as cylinders, but not numbered. The β-strands forming the DSBH-motif are coloured according to the scheme of Branden and Tooze . In the diagrams of EctD and PhyH, the disordered putative “lid” region within the segment connecting DSBH β-strands IV and V is shown by dashed lines. For the sake of clarity and comparability, DSBH β-strand II is included in the topology diagram of PhyH, although this strand is largely disordered and invisible in the corresponding crystal structure .

Figure 4. Stereo view of the EctD Fe²⁺-binding site.

A co-purified Fe³⁺ (orange sphere) is coordinated by the side chain functional groups of His-146, Asp-148 and Asp-248 of EctD and by three water molecules (shown as red spheres). The |F_obs| – |F_calc| difference electron density (blue mesh) is shown at a sigma level of 3.0 after refinement of the structural model excluding both the Fe³⁺ and its three water ligands.

Figure 5. Binding of 2-oxoglutarate by PhyH-like enzymes.

(A) PhyH, (B) SyrB2 and (C) PtlH (PDB code: 2RDN) and (D) EctD. The Fe²⁺ ion is shown as a blue sphere, water molecules are shown as red spheres. Side chains of residues involved in 2-oxoglutarate binding or in Fe²⁺ coordination are represented as sticks. Secondary structure elements are indicated as ribbons. The positioning of Phe-143 residue in the EctD structure makes in all likelihood an interaction via its aromatic side chain with 2-oxoglutarate. However, the conformation of the Phe-143 side chain shown in this figure must be regarded as tentative as it is poorly defined in the electron density map (in contrast to the Phe-143 main chain atoms). In addition, the actual orientation of the Arg-259 side chain in the EctD crystal structure is not in a position enabling its guanidino function to salt bridge the 5-carboxylate of a bound 2-oxoglutarate. However, a position allowing this interaction can easily be achieved through torsion around rotatable single bonds within the side chain of this Arg residue. It also should be noted that the DSBH β-strand II of PhyH is largely disordered in the crystal structure of this protein. In the EctD structure, the corresponding β-strand also strongly deviates from ideal β-strand geometry.

Figure 6. The ectoine hydroxylase signature sequence motif represented in the context of the EctD crystal structure.

The V. salexigens EctD structure is shown in ribbon representation with those residues constituting the EctD signature motif [F¹⁴³-X-WHSDFETWH-X-EDG-M/L-P¹⁵⁹] highlighted in yellow. This string of amino acids is invariantly present in 71 compiled EctD-type proteins (Figure S1). The side chains of the Fe²⁺-chelating amino acids (His-146, Asp-148, His-248) are shown as red sticks and those forming the putative 2-oxoglutarate-binding site (Phe-143, Ser-250, Arg-259) are represented by green sticks. The iron ligand (Fe³⁺) present in the EctD crystal structure is represented as blue sphere.