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 Fe3+ at a resolution of 1.85 Å. Like other non-heme iron(II) and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded β-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.

Different strategies of osmoadaptation in the closely related marine myxobacteria Enhygromyxa salina SWB007 and Plesiocystis pacifica SIR-1

“…Here too, the major compound was glutamate (approximately 50 %), followed by ectoine (approximately 24 %) (Schmitz & Galinski, 1996). Accumulation of both glutamate and hydroxyectoine is fully congruous with metabolism and other cellular functions (Reuter et al, 2010). However, it seems that this mechanism cannot be regarded as a general rule for myxobacteria.…”

Genetic and Biochemical Aspects of Ectoine Biosynthesis in Moderately Halophilic and Halotolerant Methylotrophic Bacteria

“…The biosynthesis of hydroxyectoine proceeds via direct hydroxylation of ectoine catalyzed by ectoine hydroxylase (EctD) (Bursy et al, 2007;Reuter et al, 2010). In Mm.…”

“…However, it does not exclude that the methanotroph can produce the bioprotectant at some environmental perturbations. NR-Database searches revealed that genomes of 67 proteobacteria and one archaeon, Nitrosopumilus maritimus, contain homologues of the ectD gene (with identities ranging from 41-55% to that of V. salexigens) (Reuter et al, 2010). ectD-like gene is either a part of the ect-cluster or it locates separately.…”

“…ectD-like gene is either a part of the ect-cluster or it locates separately. Majority of the microbial genomes contain only one copy of the ectD gene, with exception to Rhodococcus opacus B4, Marinobacter aquaeolei VT8 and C. salexigens, that possess two types of EctD-proteins (Reuter et al, 2010). In the case of C. salexigens, only one of the EctD-like enzymes contributes to the production of 5-hydroxyectoine (Garcia-Estepa et al, 2006) and the ectD gene is essential for thermoprotection of the bacterium.…”

“…The crystal structure analysis of the V. salexigens EctD (Reuter et al, 2010) showed that the folding of the protein is similar to the human phytanoyl-CoA 2-hydroxylase (McDonough et al, 2005). The core of the EctD structure consists of a double-stranded β-helix forming the main portion of the active site of the enzyme.…”