Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Abstract

By using natural abundance 13 C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine-and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctDrelated proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ect-ABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)-and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

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

“…The similar properties were observed for enzyme from Streptomyces coelicolor with respect to optimal pH (pH 7.5), temperature (32°C), K m values for ectoine (3.5 and 2.6 mM) and co-substrate 2-oxoglutarate (5.2 and 6.2 mM). Contrary to preferential production of 5-hydroxyectoine by S. coelicolor grown at 39°C (Bursy et al, 2007), the high temperature optimum of the EctD enzyme implies that there is no specific thermoactivated regulation of the hydroxylase.…”

“…The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbors the 2-oxoglutarate binding site. Despite high homology and similar reactions catalyzed by the ectoine hydroxylase and L-proline hydroxylase (Hausinger, 2004), the EctD from S. coelicolor had no additional L-proline hydroxylase activity (Bursy et al, 2007).…”

“…In various halophilic bacteria studied, the genes coding for the enzymes form a cluster ectABC (Canovas et al, 1998;Louis and Galinski, 1997;Goller et al, 1998;Kuhlmann and Bremer, 2002). Some bacteria possess an additional enzyme that converts ectoine to hydroxyectoine (ectoine hydroxylase, encoded by ectD) (Canovas et al, 1999;Prabhu et al, 2004;Bursy et al, 2007). The ect-genes are usually, but not always, organized into more or less compact operon.…”

“…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.…”

“…Ectoine hydroxylase (EctD, EC 1.14.11): Was purified from the moderate halophile Virgibacillus (Salibacillus) salexigens (Bursy et al, 2007) and from the Streptomyces coelicolor . EctD from V. salexigens is a monomeric protein of molecular mass 34 kDa being a member of the non-heme iron (II)-and 2-oxoglutarate-dependent dioxygenases.…”