Phylogenetic, biochemical and structural assessment of key enzymes in ectoine and hydroxyectoine biosynthesis

In their natural habitats, microorganisms have to frequently cope with a multitude of stressful biotic and abiotic conditions that can have adverse effects on their growth and their survival in a given ecosystem. One of the most important parameters of environmental stress due to its impact on almost all microorganisms is changing osmolarity/salinity. Among microorganisms, accumulation of compatible solutes is a widely used strategy to preserve cell integrity and growth under hyperosmotic conditions. The tetrahydropyrimidines ectoine and hydroxyectoine belong to these osmolytes and are produced by many procaryotes to minimize the adverse effects of high osmolarity on cellular physiology. Due to their beneficial impact on macromolecules, ectoine and hydroxyectoine are also reffered to in literature as chemical chaperones. These properties have spurred considerable biotechnological interest in ectoines. Ectoine is enzymatically synthesized by the ectoine synthase (EctC) and the ectoine hydroxylase (EctD) catalyses the conversion of ectoine to hydroxyectoine. Despite the fact that both enzymes have already been studied somewhat, in-depth knowledge on their phylogenetic distribution, biochemistry and structure was still lacking prior to this dissertation since only a minor number of EctC and EctD proteins has been characterized and crystal structures of both proteins containing all ligands were still missing. Since a deeper understanding of these key enzymes in ectoine biosynthesis is desirable, both with respect to basic science and industrial applications, the aim of the present dissertation was to assess the phylogenetic affiliation of ectoine biosynthetic genes and to study a selection of EctC and EctD enzymes with respect to their biochemical and kinetic properties. In addition, crystallographic approaches of EctC and EctD and site-directed mutagenesis experiments of EctD were conducted to provide a basis to unravel the position and binding motifs of the ligands within the catalytic cores of EctC and EctD. To elucidate the phylogenetic distribution of ectoine biosynthesis, the amino acid sequences of both key enzymes were used as a search query and identified, after removal of redundant sequences, about 723 potential ectoine producers of which only 12 originated from Archaea. This analysis revealed that ectoine biosynthesis is widely distributed in prokaryotes, predominantly in members of the Bacteria, underlining the important role of ectoines in microbial stress responses. On this basis, various ectoine synthases and ectoine hydroxylases deriving from different organisms have been biochemically and/or kinetically characterized that were widely distributed on the phylogenetic tree of ectoine biosynthesis. Each of the so far studied proteins possessed similar enzyme kinetics, however, comparison of their biochemical characteristics revealed only minor differences between EctD proteins but major variations between EctC enzymes. This suggests that the ectoine synthase, whose properties partially reflect the environmental circumstances of their hosts, might have developed in terms of evolution prior to the ectoine hydroxylase. Identification of EctC and EctD proteins possessing increased stability allowed new crystallization trials. Multiple crystal structures have been solved in the course of this dissertation in collaboration with Dr. Sander Smits (University of Düsseldorf). In terms of EctD, structures in its apo-form, in complex with the iron co-factor and in complex with the iron catalyst, the co-substrate 2-oxoglutarate and the reaction product hydroxyectoine have been solved. These structures provided, in connection with comprehensive site-directed mutagenesis experiments, a detailed view into the catalytic core of EctD allowing a proposal for its catalyzed reaction mechanisms (Proposal by Dr. Wolfgang Buckel). In terms of EctC, a detailed expression and purification protocol for the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis has been described in collaboration with Dr. Sander Smits (University of Düsseldorf) that identifies EctC to form dimers in solution and deals with crystallization trials and preliminary X-ray diffraction data providing a promising basis for the solution of its crystal structure, which has, based on the presented data, been published posterior to the present dissertation. Collectively, the present dissertation provides detailed information about the phylogenetic distribution and biochemistry of EctC and EctD as well as the solved crystal structure of the ectoine hydroxylase, the key enzymes in hydroxyectoine biosynthesis, and promising crystallization trials of EctC, the key enzyme in ectoine biosynthesis, and thus substantially contributes to the understanding of the role of ectoines in the global microbial osmostress adaptation

Phylogenetic, biochemical and structural assessment of key enzymes in ectoine and hydroxyectoine biosynthesis

In their natural habitats, microorganisms have to frequently cope with a multitude of stressful biotic and abiotic conditions that can have adverse effects on their growth and their survival in a given ecosystem. One of the most important parameters of environmental stress due to its impact on almost all microorganisms is changing osmolarity/salinity. Among microorganisms, accumulation of compatible solutes is a widely used strategy to preserve cell integrity and growth under hyperosmotic conditions. The tetrahydropyrimidines ectoine and hydroxyectoine belong to these osmolytes and are produced by many procaryotes to minimize the adverse effects of high osmolarity on cellular physiology. Due to their beneficial impact on macromolecules, ectoine and hydroxyectoine are also reffered to in literature as chemical chaperones. These properties have spurred considerable biotechnological interest in ectoines. Ectoine is enzymatically synthesized by the ectoine synthase (EctC) and the ectoine hydroxylase (EctD) catalyses the conversion of ectoine to hydroxyectoine. Despite the fact that both enzymes have already been studied somewhat, in-depth knowledge on their phylogenetic distribution, biochemistry and structure was still lacking prior to this dissertation since only a minor number of EctC and EctD proteins has been characterized and crystal structures of both proteins containing all ligands were still missing. Since a deeper understanding of these key enzymes in ectoine biosynthesis is desirable, both with respect to basic science and industrial applications, the aim of the present dissertation was to assess the phylogenetic affiliation of ectoine biosynthetic genes and to study a selection of EctC and EctD enzymes with respect to their biochemical and kinetic properties. In addition, crystallographic approaches of EctC and EctD and site-directed mutagenesis experiments of EctD were conducted to provide a basis to unravel the position and binding motifs of the ligands within the catalytic cores of EctC and EctD. To elucidate the phylogenetic distribution of ectoine biosynthesis, the amino acid sequences of both key enzymes were used as a search query and identified, after removal of redundant sequences, about 723 potential ectoine producers of which only 12 originated from Archaea. This analysis revealed that ectoine biosynthesis is widely distributed in prokaryotes, predominantly in members of the Bacteria, underlining the important role of ectoines in microbial stress responses. On this basis, various ectoine synthases and ectoine hydroxylases deriving from different organisms have been biochemically and/or kinetically characterized that were widely distributed on the phylogenetic tree of ectoine biosynthesis. Each of the so far studied proteins possessed similar enzyme kinetics, however, comparison of their biochemical characteristics revealed only minor differences between EctD proteins but major variations between EctC enzymes. This suggests that the ectoine synthase, whose properties partially reflect the environmental circumstances of their hosts, might have developed in terms of evolution prior to the ectoine hydroxylase. Identification of EctC and EctD proteins possessing increased stability allowed new crystallization trials. Multiple crystal structures have been solved in the course of this dissertation in collaboration with Dr. Sander Smits (University of Düsseldorf). In terms of EctD, structures in its apo-form, in complex with the iron co-factor and in complex with the iron catalyst, the co-substrate 2-oxoglutarate and the reaction product hydroxyectoine have been solved. These structures provided, in connection with comprehensive site-directed mutagenesis experiments, a detailed view into the catalytic core of EctD allowing a proposal for its catalyzed reaction mechanisms (Proposal by Dr. Wolfgang Buckel). In terms of EctC, a detailed expression and purification protocol for the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis has been described in collaboration with Dr. Sander Smits (University of Düsseldorf) that identifies EctC to form dimers in solution and deals with crystallization trials and preliminary X-ray diffraction data providing a promising basis for the solution of its crystal structure, which has, based on the presented data, been published posterior to the present dissertation. Collectively, the present dissertation provides detailed information about the phylogenetic distribution and biochemistry of EctC and EctD as well as the solved crystal structure of the ectoine hydroxylase, the key enzymes in hydroxyectoine biosynthesis, and promising crystallization trials of EctC, the key enzyme in ectoine biosynthesis, and thus substantially contributes to the understanding of the role of ectoines in the global microbial osmostress adaptation

electronic reprint Overproduction, crystallization and X-ray diffraction data analysis of ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis IUCr Journals CRYSTALLOGRAPHY JOURNALS ONLINE Overproduction, crystallization and X-r

Ectoine biosynthetic genes (ectABC) are widely distributed in bacteria. Microorganisms that carry them make copious amounts of ectoine as a cell protectant in response to high-osmolarity challenges. Ectoine synthase (EctC; EC 4.2.1.108) is the key enzyme for the production of this compatible solute and mediates the last step of ectoine biosynthesis. It catalyzes the ring closure of the cyclic ectoine molecule. A codon-optimized version of ectC from Sphingopyxis alaskensis (Sa) was used for overproduction of SaEctC protein carrying a Streptag II peptide at its carboxy-terminus. The recombinant SaEctC-Strep-tag II protein was purified to near-homogeneity from Escherichia coli cell extracts by affinity chromatography. Size-exclusion chromatography revealed that it is a dimer in solution. The SaEctC-Strep-tag II protein was crystallized using the sitting-drop vapour-diffusion method and crystals that diffracted to 1.0 Å resolution were obtained

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-β-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

Salt-sensitivity of σHand Spo0A prevents sporulation of Bacillus subtilisat high osmolarity avoiding death during cellular differentiation

The spore-forming bacterium Bacillus subtilis frequently experiences high osmolarity as a result of desiccation in the soil. The formation of a highly desiccation-resistant endospore might serve as a logical osmostress escape route when vegetative growth is no longer possible. However, sporulation efficiency drastically decreases concomitant with an increase in the external salinity. Fluorescence microscopy of sporulation-specific promoter fusions to gfp revealed that high salinity blocks entry into the sporulation pathway at a very early stage. Specifically, we show that both Spo0A- and SigH-dependent transcription are impaired. Furthermore, we demonstrate that the association of SigH with core RNA polymerase is reduced under these conditions. Suppressors that modestly increase sporulation efficiency at high salinity map to the coding region of sigH and in the regulatory region of kinA, encoding one the sensor kinases that activates Spo0A. These findings led us to discover that B. subtilis cells that overproduce KinA can bypass the salt-imposed block in sporulation. Importantly, these cells are impaired in the morphological process of engulfment and late forespore gene expression and frequently undergo lysis. Altogether our data indicate that B. subtilis blocks entry into sporulation in high-salinity environments preventing commitment to a developmental program that it cannot complete

Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily.

Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel

Biochemical properties of the studied EctD-type proteins.

<p>The biochemical properties of the studied EctD-type proteins were determined as described in Material and Methods. The given temperature, pH and salt ranges describe a window in which the tested enzymes still exhibited some degree of activity.</p

Genetic organization of the ectoine/hydroxyectoine biosynthesis gene clusters.

<p>The different types of <i>ect</i> gene clusters present in putative ectoine/hydroxyectoine producers are represented. An example for the genetic organization of each type of <i>ect</i> cluster found in the <i>ectC</i> reference set (440 representatives; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093809#pone-0093809-g001" target="_blank">Fig. 1</a>) is given along with a microorganism in which it occurs. (A) Most common organizational types of the <i>ect</i> gene clusters. (B) Representatives of the organization of the ectoine/hydroxyectoine biosynthetic genes that deviate from the otherwise commonly found genetic organization.</p

Phylogenetic tree of EctC- and EctD-type proteins.

<p>The shown phylogenetic tree is based on the alignment of EctC amino acid sequences identified by a BLAST search at the JGI Web-server that were then aligned using ClustalW. These compiled amino acid sequences were then used to assess the phylogenetic distribution of the EctC protein using the iTOL Web-server. Evolutionary distances are not given. The color code indicates the distribution of EctC among members of the <i>Bacteria</i> and <i>Archaea</i>. The presence of an <i>ectD</i> gene in a given microbial species possessing <i>ectC</i> is indicated by black (<i>ectD</i> is part of the <i>ect</i> gene cluster) or red circles (<i>ectD</i> is located outside of the <i>ect</i> gene cluster). Purple circles are indicating the presence of an <i>ask_ect</i> gene associated with the <i>ect</i> gene cluster, whereas the presence of an <i>ectR</i> regulatory gene is indicted by green circles. If different strains of the same species were sequenced, only one representative symbolizes them. For instance, there are genomic data of 139 strain of <i>Vibrio cholerae</i> available in the database, each of which possesses an <i>ectABC</i> gene cluster, but only one of these sequences was used for the phylogenetic analysis.</p