Arsenobetaine: an ecophysiologically important organoarsenical confers cytoprotection against osmotic stress and growth temperature extremes.

Arsenic, a highly cytotoxic and cancerogenic metalloid, is brought into the biosphere through geochemical sources and anthropogenic activities. A global biogeochemical arsenic biotransformation cycle exists in which inorganic arsenic species are transformed into organoarsenicals, which are subsequently mineralized again into inorganic arsenic compounds. Microorganisms contribute to this biotransformation process greatly and one of the organoarsenicals synthesized and degraded in this cycle is arsenobetaine. Its nitrogen-containing homologue glycine betaine is probably the most frequently used compatible solute on Earth. Arsenobetaine is found in marine and terrestrial habitats and even in deep-sea hydrothermal vent ecosystems. Despite its ubiquitous occurrence, the biological function of arsenobetaine has not been comprehensively addressed. Using Bacillus subtilis as a well-understood platform for the study of microbial osmostress adjustment systems, we ascribe here to arsenobetaine both a protective function against high osmolarity and a cytoprotective role against extremes in low and high growth temperatures. We define a biosynthetic route for arsenobetaine from the precursor arsenocholine that relies on enzymes and genetic regulatory circuits for glycine betaine formation from choline, identify the uptake systems for arsenobetaine and arsenocholine, and describe crystal structures of ligand-binding proteins from the OpuA and OpuB ABC transporters complexed with either arsenobetaine or arsenocholine.

Strangers in the archaeal world: osmostress-responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus.

Ectoine and hydroxyectoine are compatible solutes widely synthesized by members of the Bacteria to cope with high osmolarity surroundings. Inspection of 557 archaeal genomes revealed that only 12 strains affiliated with the Nitrosopumilus, Methanothrix or Methanobacterium genera harbour ectoine/hydroxyectoine gene clusters. Phylogenetic considerations suggest that these Archaea have acquired these genes through horizontal gene transfer events. Using the Thaumarchaeon 'Candidatus Nitrosopumilus maritimus' as an example, we demonstrate that the transcription of its ectABCD genes is osmotically induced and functional since it leads to the production of both ectoine and hydroxyectoine. The ectoine synthase and the ectoine hydroxylase were biochemically characterized, and their properties resemble those of their counterparts from Bacteria. Transcriptional analysis of osmotically stressed 'Ca. N. maritimus' cells demonstrated that they possess an ectoine/hydroxyectoine gene cluster (hyp-ectABCD-mscS) different from those recognized previously since it contains a gene for an MscS-type mechanosensitive channel. Complementation experiments with an Escherichia coli mutant lacking all known mechanosensitive channel proteins demonstrated that the (Nm)MscS protein is functional. Hence, 'Ca. N. maritimus' cells cope with high salinity not only through enhanced synthesis of osmostress-protective ectoines but they already prepare themselves simultaneously for an eventually occurring osmotic down-shock by enhancing the production of a safety-valve (NmMscS).

A specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501.

The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. l-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. l-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from l-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an askgene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_askgenes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. colistrain carrying the plasmid-based ectABCD_askgene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCDgene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis.

Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stresses.

Streptomyces coelicolor A3(2) synthesizes ectoine and 5-hydroxyectoine upon the imposition of either salt (0.5 M NaCl) or heat stress (39 degrees C). The cells produced the highest cellular levels of these compatible solutes when both stress conditions were simultaneously imposed. Protection against either severe salt (1.2 M NaCl) or heat stress (39 degrees C) or a combination of both environmental cues could be accomplished by adding low concentrations (1 mM) of either ectoine or 5-hydroxyectoine to S. coelicolor A3(2) cultures. The best salt and heat stress protection was observed when a mixture of ectoine and 5-hydroxyectoine (0.5 mM each) was provided to the growth medium. Transport assays with radiolabeled ectoine demonstrated that uptake was triggered by either salt or heat stress. The most effective transport and accumulation of [(14)C]ectoine by S. coelicolor A3(2) were achieved when both environmental cues were simultaneously applied. Our results demonstrate that the accumulation of the compatible solutes ectoine and 5-hydroxyectoine allows S. coelicolor A3(2) to fend off the detrimental effects of both high salinity and high temperature on cell physiology. We also characterized the enzyme (EctD) required for the synthesis of 5-hydroxyectoine from ectoine, a hydroxylase of the superfamily of the non-heme-containing iron(II)- and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). The gene cluster (ectABCD) encoding the enzymes for ectoine and 5-hydroxyectoine biosynthesis can be found in the genome of S. coelicolor A3(2), Streptomyces avermitilis, Streptomyces griseus, Streptomyces scabiei, and Streptomyces chrysomallus, suggesting that these compatible solutes play an important role as stress protectants in the genus Streptomyces.

Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms.

Ectoine and 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 from the precursor L-aspartate-ß-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo-form, thereby revealing that the iron-free structure exists already in a pre-set configuration to incorporate the iron catalyst. Collectively, our work defines the taxonomic distribution and salient biochemical properties of the ectoine hydroxylase protein family and contributes to the understanding of its structure.

Molecular dynamics simulations and structure-guided mutagenesis provide insight into the architecture of the catalytic core of the ectoine hydroxylase.

Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.

The crystal structure of the substrate-binding protein OpuBC from Bacillus subtilis in complex with choline.

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K(d) value of about 30 µM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.

Arg149 is involved in switching the low affinity, open state of the binding protein AfProX into its high affinity, closed state.

The substrate binding protein AfProX from the Archaeoglobus fulgidus ProU ATP binding cassette transporter is highly selective for the compatible solutes glycine betaine (GB) and proline betaine, which confer thermoprotection to this hyperthermophilic archaeon. A detailed mutational analysis of the substrate binding site revealed the contribution of individual amino acids for ligand binding. Replacement of Arg149 by an Ala residue displayed the largest impact on substrate binding. The structure of a mutant AfProX protein (substitution of Tyr111 with Ala) in complex with GB was solved in the open liganded conformation to gain further insight into ligand binding. In this crystal structure, GB is bound differently compared to the GB closed liganded structure of the wild-type AfProX protein. We found that a network of amino acid side chains communicates the presence of GB toward Arg149, which increases ligand affinity and induces domain closure of AfProX. These results were corroborated by molecular dynamics studies and support the view that Arg149 finalizes the high-affinity state of the AfProX substrate binding protein.

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

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.

The crystal structure of UehA in complex with ectoine-A comparison with other TRAP-T binding proteins.

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.