Halothermothrix Orenii Research Articles

The amino acid composition of proteins from anaerobic halophilic bacteria of the order Halanaerobiales

We performed a comparative analysis of the genome sequences of three anaerobic halophilic fermentative bacteria belonging to the order Halanaerobiales: Halanaerobium praevalens, the alkaliphilic "Halanaerobium hydrogeniformans", and the thermophilic Halothermothrix orenii to assess the amino acid composition of their proteins. Members of the Halanaerobiales were earlier shown to accumulate KCl rather than organic compatible solutes for osmotic balance, and therefore the presence of a dominantly acidic proteome was predicted. Past reports indeed showed a large excess of acidic over basic amino acids in whole-cell hydrolysates of selected members of the order. However, the genomic analysis did not show unusually high contents of acidic amino acids or low contents of basic amino acids. The apparent excess of acidic amino acids in these anaerobic halophiles reported earlier is due to the high content in their proteins of glutamine and asparagine, which yield glutamate and aspartate upon acid hydrolysis. It is thus suggested that the proteins of the Halanaerobiales, which are active in the presence of high intracellular KCl concentrations, do not possess the typical acidic signature of the 'halophilic' proteins of the Archaea of the order Halobacteriales or of the extremely halophilic bacterium Salinibacter.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophile Halothermothrix orenii.

A ribokinase gene (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and overexpressed in Escherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 45.6, b = 61.1, c = 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

Functional characterization of Class II 5-enopyruvylshikimate-3-phosphate synthase from Halothermothrix orenii H168 in Escherichia coli and transgenic Arabidopsis

Although a large number of AroA enzymes (5-enopyruvylshikimate-3-phosphate synthase [EPSPS]) have been identified, cloned and tested for glyphosate resistance, only AroA variants derived from Agrobacterium tumefaciens strain CP4 have been successfully used commercially. We have now used a polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method to synthesize an aroA gene (aroA(H. orenii)) from Halothermothrix orenii H168 encoding a new EPSPS similar to AroA(A. tumefaciens CP4.) AroA(H. orenii) was then expressed in Escherichia coli and key kinetic values of the purified enzyme were determined. Kinetic analysis of AroA(H. orenii) indicated that the full-length enzyme exhibited increased tolerance to glyphosate compared with E. coli AroA(E. coli) while retaining a high affinity for the substrate phosphoenolpyruvate. Transgenic Arabidopsis plants containing aroA(H. orenii) were resistant to 15 mM glyphosate. Site-directed mutagenesis showed that residues Thr355Ser affected the affinity of AroA(H. orenii) for glyphosate, providing further evidence that specific amino acid residues are responsible for differences in enzymatic behavior among different AroA enzymes.

Expression, purification and preliminary crystallographic analysis of the recombinant β-glucosidase (BglA) from the halothermophile Halothermothrix orenii.

The β-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress.

Crystal structure of a fructokinase homolog from Halothermothrix orenii

Fructokinase (FRK; EC 2.7.1.4) catalyzes the phosphorylation of d-fructose to d-fructose 6-phosphate (F6P). This irreversible and near rate-limiting step is a central and regulatory process in plants and bacteria, which channels fructose into a metabolically active state for glycolysis. Towards understanding the mechanism of FRK, here we report the crystal structure of a FRK homolog from a thermohalophilic bacterium Halothermothrixorenii (Hore_18220 in sequence databases). The structure of the Hore_18220 protein reveals a catalytic domain with a Rossmann-like fold and a beta-sheet "lid" for dimerization. Based on comparison of Hore_18220 to structures of related proteins, we propose its mechanism of action, in which the lid serves to regulate access to the substrate binding sites. Close relationship of Hore_18220 and plant FRK enzymes allows us to propose a model for the structure and function of FRKs.

The Structure of Sucrose Phosphate Synthase from Halothermothrix orenii Reveals Its Mechanism of Action and Binding Mode

Sucrose phosphate synthase (SPS) catalyzes the transfer of a glycosyl group from an activated donor sugar, such as uridine diphosphate glucose (UDP-Glc), to a saccharide acceptor D-fructose 6-phosphate (F6P), resulting in the formation of UDP and D-sucrose-6'-phosphate (S6P). This is a central regulatory process in the production of sucrose in plants, cyanobacteria, and proteobacteria. Here, we report the crystal structure of SPS from the nonphotosynthetic bacterium Halothermothrix orenii and its complexes with the substrate F6P and the product S6P. SPS has two distinct Rossmann-fold domains with a large substrate binding cleft at the interdomain interface. Structures of two complexes show that both the substrate F6P and the product S6P bind to the A-domain of SPS. Based on comparative analysis of the SPS structure with other related enzymes, the donor substrate, nucleotide diphosphate glucose, binds to the B-domain of SPS. Furthermore, we propose a mechanism of catalysis by H. orenii SPS. Our findings indicate that SPS from H. orenii may represent a valid model for the catalytic domain of plant SPSs and thus may provide useful insight into the reaction mechanism of the plant enzyme.

Crystal Structure of the Polyextremophilic α-Amylase AmyB from Halothermothrix orenii: Details of a Productive Enzyme–Substrate Complex and an N Domain with a Role in Binding Raw Starch

The gene for a membrane-bound, halophilic, and thermostable α-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain ( N domain) that forms a large groove—the N– C groove—some 30 Å away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 Å resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with α-cyclodextrin and maltoheptaose at 2.2 Å resolution, a maltotetraose molecule is bound in subsites − 1 to + 3, spanning the cleavage point at − 1/+ 1, with the − 1 glucosyl residue present as a 2S o skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across − 1/+ 1 and ring distortion. We observe three methionine side chains that serve as “binding platforms” for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N– C groove can accommodate, spatially and chemically, large substrates such as A-starch.

Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly-extreme condition

Here we report the first crystal structure of a protein, AmyA, a secretory α-amylase isolated from Halothermothrix orenii, which is both halophilic and thermophilic. The crystal structure was determined at 1.6 Å resolution. AmyA lacks the conserved acidic surface, which is considered essential for protein stability at high salinity. Sedimentation velocity and CD experiments on AmyA reveal the formation of unique reversible poly-dispersed oligomers that show unusually high thermal stability. These studies provide valuable insight into the structural elements that contribute to the stability of AmyA at both physical and chemical extremes and their functional implications.

Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) fromHalothermothrix orenii

This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 A, beta = 103.16 degrees, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 A. Heavy-metal and halide-soaking trials are currently in progress to solve the structure.

Crystallization of a novel alpha-amylase, AmyB, from the thermophilic halophile Halothermothrix orenii.

This is a report on the structure determination of AmyB, the second alpha-amylase from Halothermothrix orenii, by X-ray crystallography. This bacterium was isolated from saltpans where conditions consisted of both high temperatures and high NaCl content. AmyB is a 599-residue protein which is stable and significantly active at 358 K in starch solution containing up to 10%(w/v) NaCl. The purified recombinant AmyB protein crystallizes in the monoclinic space group C2, with unit-cell parameters a = 225.85, b = 77.16, c = 50.13 A, beta = 99.32 degrees, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 1.97 A.

Crystallization of an alpha-amylase, AmyA, from the thermophilic halophile Halothermothrix orenii.

This report is the first crystallographic study of an amylase from an organism that is both thermophilic and halophilic. alpha-Amylase from the thermophilic halophile Halothermothrix orenii (AmyA) is a 515-residue protein. It is stable and significantly active at 338 K in starch solution containing NaCl [up to 25%(w/v)]. Purified recombinant AmyA protein crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.126, b = 61.658, c = 147.625 A, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 1.89 A.

Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme.

A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA. The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the alpha-amylase family and showed the greatest homology to the Bacillus megaterium group of alpha-amylases. The amyA gene was expressed in E. coli as a hexahistidine-tagged enzyme and purified. The purified recombinant enzyme was optimally active at 65 degrees C in 5% (w/v) NaCl at pH 7.5, with significant activity retained in the presence of up to 25% (w/v) NaCl. It had a specific activity of 22.32 U mg(-1) and required NaCl and CaCl(2) for optimum activity and thermostability. The relatively high proportion of acidic amino acids typically observed for many enzymes from halophiles was absent in H. orenii AmyA.

Random sequence analysis of genomic DNA of an anaerobic, thermophilic, halophilic bacterium, Halothermothrix orenii.

A pBluescriptSK+ vector library consisting of 3.360 clones with an average insert size of 3.5 kb was constructed from the genome of Halothermothrix orenii, a halophilic and thermoanaerobic member of the family Haloanaerobiaceae. From both ends, 77 clones were sequenced using T3 and T7 vector primers generating 154 sequence tags, representing approximately 85 kb of the genome. Comparison of sequence tags against the Gen-Bank database using BLASTX identified 66 known proteins and 15 conserved hypothetical proteins. The putative proteins included a V-ATPase, hydrogenases, and enzymes with potential for industrial applications. The overall G + C% of the codons used was 42.9% with a third-position G + C content of 38.6%. High levels of excess acidic amino acids were not detected in the putative proteins of H. orenii as compared to the mesophilic haloanaerobes. This lack may be the result of reduced activity of acidic, halophilic enzymes at high temperatures and intermediate salt concentrations.