Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases

Abstract

The Fe(II)/αketoglutarate (αKG) dependent oxygenases catalyze a diverse range of reactions significant in biological processes such as antibiotic biosynthesis, lipid metabolism, oxygen sensing, and DNA and RNA repair. Although functionally diverse, the eight-stranded β-barrel (cupin) and HX(D/E)XnH facial triad motifs are conserved in this super-family of enzymes. Crystal structure analysis of 25 αKG oxygenases reveals two stereoisomers of the Fe cofactor, Anti and Clock, which differ in the relative position of the exchangeable ligand position and the primary substrate. Herein, we discuss the relationship between the chemical mechanism and the secondary coordination sphere of the αKG oxygenases, within the constraints of the stereochemistry of the Fe cofactor. Sequence analysis of the cupin barrel indicates that a small subset of positions constitute the second coordination sphere, which has significant ramifications for the structure of the ferryl intermediate. The competence of both Anti and Clock stereoisomers of Fe points to a ferryl intermediate that is 5 coordinate. The small number of conserved close contacts within the active sites of αKG oxygenases can be extended to chemically related enzymes, such as the αKG-dependent halogenases SyrB2 and CytC3, and the non-αKG dependent dioxygenases isopenicillin N synthase (IPNS) and cysteine dioxygenase (CDO).

Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site

“…. H motif, the so-called 2-His-1-carboxylate facial triad, forming a type of mononuclear iron center found in many members of the dioxygenase superfamily (37)(38)(39)(40)64). The three iron-binding residues are fully conserved among a group of 433 aligned EctD-type proteins (22).…”

“…4A). This core, also known as the jelly roll or cupin fold (38,39), is formed by two four-stranded anti-parallel ␤-sheets that are arranged in the form of a ␤-sandwich (Fig. 4A).…”

“…The ectoine hydroxylase is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily, a group of versatile biocatalysts that not only carry out hydroxylation reactions but also dimethylations, desaturations, cyclizations, ring expansions, epimerizations, and halogenations (37)(38)(39)(40)(41). Although the type of substrates used by these dioxygenases varies considerably, common enzyme reaction mechanisms are observed.…”

“…Although the type of substrates used by these dioxygenases varies considerably, common enzyme reaction mechanisms are observed. Most members of this superfamily couple the two-electron oxidation of their substrates with the reaction of oxygen and 2-oxoglutarate (38,39,42). The ectoine hydroxylase adheres to this general reaction scheme (27,43).…”

“…Despite their varied enzyme activities, non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenases are structurally closely related and typically consist of a double-stranded ␤-helix (DSBH) 5 core, the so-called jelly roll or cupin fold (37)(38)(39)(40). Crystallographic analysis of the ectoine hydroxylase from the salt-tolerant moderate halophile Virgibacillus salexigens (Vs) in its iron-bound (44) and iron-free (22) forms revealed that the DSBH in the VsEctD protein is formed by fourstranded antiparallel ␤-sheets arranged in form of a ␤-sandwich; it is decorated with and stabilized by a number of ␣-helices (22,44).…”