The Ectoine Hydroxylase: A Nonheme-Containing Iron(II) and 2-Oxoglutarate-Dependent Dioxygenase

Ectoine and 5-hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction catalyzed by the EctD protein, a member of the nonheme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. Structures of EctD are known from the Virgibacillus salexigens EctD protein in its apo- and iron-bound forms. Furthermore, a set of crystal structures is known from the EctD protein from Sphingopyxis alaskensis in its apo- and Fe(II)-bound forms and also from a dead-end complex of EctD together with the substrate 2-oxogluterate and the product 5-hydroxyectoine. In this article, we describe the purification and biochemically characterization of nine different EctD proteins from different species. Furthermore, the importance of the Fe(II) ligand and its binding site is described. The octahedral geometry of Fe(II) binding is mediated via two histidine and one aspartate side chain together with water molecules. The latter are replaced by the binding of the 2-oxoglutarate substrate to the EctD enzyme. The binding mechanism of Fe(II) is strictly conserved within the EctD protein superfamily. 3D Structure Structure of the dimeric ectoine hydroxylase from S. alaskensis (SaEctD). One monomer is represented as cartoon representation. The other monomer is highlighted as a surface with the bound 2-oxoglutarate substrate (shown as blue sticks) and the 5-hydroxyectoine product (shown in orange). The bound Fe(II) ligand is shown as spheres colored in magenta (PDB code: 4Q5O). The signature sequence motif of SaEctD is colored in green. These figures were prepared using PyMOL (www.pymol.org). Keywords: compatible solutes; osmostress protectants; cupin; mononuclear iron center