Thermobaculum Terrenum Research Articles

Structural characterization of the type I-B CRISPR Cas7 from Thermobaculum terrenum

Clustered regularly interspaced short palindromic repeats (CRISPR) in many prokaryotes functions as an adaptive immune system against mobile genetic elements. A heterologous ribonucleoprotein silencing complex composed of CRISPR-associated (Cas) proteins and a CRISPR RNA (crRNA) neutralizes the incoming mobile genetic elements. The type I and III silencing complexes commonly include a protein-helical backbone of several copies of identical subunits, for example, Cas7 in the type I silencing complex.In this study, we structurally characterized type I-B Cas7 (Csh2 from Thermobaculum terrenum; TterCsh2). The revealed crystal structure of TterCsh2 shows a typical glove-like architecture of Cas7, which consists of a palm, a thumb, and a finger domain. Csh2 proteins have 5 conserved sequence motifs that are arranged to form a presumable crRNA-binding site in the TterCsh2 structure. This crRNA binding site of TterCsh2 is structurally and potentially comparable to those observed in helix-forming Cas7 structures in other sub-types. Analysis of the reported Cas7 structures and their sequences suggests that Cas7s can be divided into at least two sub-classes. These data will broaden our understanding on the Cascade complex of CRISPR/Cas systems.

Counterbalance of Stability and Activity Observed for Thermostable Transaminase from Thermobaculum terrenum in the Presence of Organic Solvents

Pyridoxal-5’-phosphate-dependent transaminases catalyze stereoselective amination of organic compounds and are highly important for industrial applications. Catalysis by transaminases often requires organic solvents to increase the solubility of reactants. However, natural transaminases are prone to inactivation in the presence of water-miscible organic solvents. Here, we present the solvent tolerant thermostable transaminase from Thermobaculum terrenum (TaTT) that catalyzes transamination between L-leucine and alpha-ketoglutarate with an optimum at 75 °C and increases the activity ~1.8-fold upon addition of 15% dimethyl sulfoxide or 15% methanol at high but suboptimal temperature, 50 °C. The enhancement of the activity correlates with a decrease in the thermal denaturation midpoint temperature. The blue-shift of tryptophan fluorescence suggested that solvent molecules penetrate the hydration shell of the enzyme. Analysis of hydrogen bonds in the TaTT dimer revealed a high number of salt bridges and surface hydrogen bonds formed by backbone atoms. The latter are sensitive to the presence of organic solvents; they rearrange, conferring the relaxation of some constraints inherent to a thermostable enzyme at low temperatures. Our data support the idea that the counterbalance of stability and activity is crucial for the catalysis under given conditions; the obtained results may be useful for fine-tuning biocatalyst efficiency.

Dodecamers derived from the crystal structure were found in the pre-crystallization solution of the transaminase from the thermophilic bacterium Thermobaculum terrenum by small-angle X-ray scattering

The pre-crystallization solution of the transaminase from Thermobaculum terrenum (TaTT) has been studied by small-angle X-ray scattering (SAXS). Regular changes in the oligomeric composition of the protein were observed after the addition of the precipitant. Comparison of the observed oligomers with the crystal structure of TaTT (PDB ID 6GKR) shows that dodecamers may act as building blocks in the growth of transaminase single crystals. Correlating of these results to the similar X-ray studies of other proteins suggests that SAXS may be a valuable tool for searching optimum crystallization conditions.AbbreviationSAXSsmall-angle X-ray scatteringTatransaminaseTaTTtransaminase from Thermobaculum terrenumPLPpyridoxal-5’-phosphateR-PEAR-(þ)-1-phenylethylamineBCATbranched-chain amino acid aminotransferaseDAATD-aminoacid aminotransferaseR-TAR-amine:pyruvate transaminaseCommunicated by Ramaswamy H. Sarma

Computational and Enzymatic Analyses Unveil the Catalytic Mechanism of Thermostable Trehalose Synthase and Suggest Strategies for Improved Bioconversion.

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, and is therefore essential for trehalose production. Consequently, dissecting the catalytic mechanism of TreS is important for enzyme optimization and industrial applications. TreS from Thermobaculum terrenum (TtTreS) is a thermostable enzyme. Here, we studied the composition of the TtTreS active site through computer calculation and enzyme analysis. The results were consistent with a two-step double-displacement mechanism, similar to that of glycoside hydrolase 13 family enzymes. However, our data suggested that glucose rotation, following breakage of the α-1,4 glycosidic bond, is a key factor determining the reaction direction and conversion rate. The N246 residue plays an important role in glucose rotation. Moreover, we established a saturated mutation model for the nonconserved amino acids around the substrate gateway domain. Finally, four TtTreS mutants (K136T, Y137D, K138N, and D139S) resulted in improved trehalose yield compared to that of the wild-type enzyme.

Biochemical and structural insights into PLP fold type IV transaminase from Thermobaculum terrenum

The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task. Here we report a detailed structural and kinetic study of thermostable transaminase from the bacterium Thermobaculum terrenum (TaTT) using the methods of enzyme kinetics, X-ray crystallography and molecular modeling. TaTT can convert L-branched-chain and L-aromatic amino acids as well as (R)-(+)-1-phenylethylamine at a high rate and with high enantioselectivity. The structures of TaTT in complex with the cofactor pyridoxal 5′-phosphate covalently bound to enzyme and in complex with its reduced form, pyridoxamine 5′-phosphate, were determined at resolutions of 2.19 Å and 1.5 Å, and deposited in the Protein Data Bank as entries 6GKR and 6Q8E, respectively. TaTT is a fold type IV PLP-dependent enzyme. In terms of structural similarity, the enzyme is close to known branched-chain amino acid aminotransferases, but differences in characteristic sequence motifs in the active site were observed in TaTT compared to canonical branched-chain amino acid aminotransferases, which can explain the improved binding of aromatic amino acids and (R)-(+)-1-phenylethylamine. This study has shown for the first time that high substrate specificity towards both various l-amino acids and (R)-primary amines can be implemented within one pyridoxal 5′-phosphate-dependent active site of fold type IV. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further biochemical and bioinformatic studies to understand the sequence-structure-function relationship in these enzymes.

Combination of sequence-based and in silico screening to identify novel trehalose synthases

A combined approach of sequence-based screening from metagenomic soil DNA and subsequent in silico screening was established to identify novel trehalose synthases (TS, EC 5.4.99.16). Metagenomic DNA was isolated from diverse soil samples and used as template for PCR-based screening targeted against conserved regions of trehalose synthases. This resulted in four metagenomic TS-like fragments with broad sequence diversity (41−67% identity to each other). The encoded open reading frames were used as templates for further in silico screening. Two trehalose synthases were discovered using this novel approach and their enzymatic properties were further investigated. The trehalose synthase from Micrococcus terreus MtTS exhibited a broad pH optimum between 6.5 and 7.5 with highest reaction velocity at 35 °C and a protruding stability at this temperature (t1/2 = 50 h). Characteristic of enzymes from thermophilic organisms, the trehalose synthase from Thermobaculum terrenum had a distinct temperature optimum at 50 °C, exhibiting also a prominent half time with t1/2 = 45 h at pH 6.5. Both bioconversions resulted in final trehalose levels of 60%, whereas TtTS produced reduced amounts of the byproduct glucose (10%) compared with MtTS (15%), which is favorable for trehalose production. This combined screening approach intended to circumvent the bottleneck of metagenomic enzyme mining, regarding time and cost of intensive screening procedures for industrial relevant biocatalysts such as trehalose synthases.

Structural Characteristics and Function of a New Kind of Thermostable Trehalose Synthase from Thermobaculum terrenum.

Trehalose has important applications in the food industry and pharmaceutical manufacturing. The thermostable enzyme trehalose synthase from Thermobaculum terrenum (TtTS) catalyzes the reversible interconversion of maltose and trehalose. Here, we investigated the structural characteristics of TtTS in complex with the inhibitor TriS. TtTS exhibits the typical three domain glycoside hydrolase family 13 structure. The catalytic cleft consists of Asp202-Glu244-Asp310 and various conserved substrate-binding residues. However, among trehalose synthases, TtTS demonstrates obvious thermal stability. TtTS has more polar (charged) amino acids distributed on its protein structure surface and more aromatic amino acids buried within than other mesophilic trehalose synthases. Furthermore, TtTS structural analysis revealed four potential metal ion-binding sites rather than the two in a homologous structure. These factors may render TtTS relatively more thermostable among mesophilic trehalose synthases. The detailed thermophilic enzyme structure provided herein may provide guidance for further protein engineering in the design of stabilized enzymes.

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum

Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system.

TtMCO: A highly thermostable laccase-like multicopper oxidase from the thermophilic Thermobaculum terrenum

This paper reports the identification, heterologous expression in Escherichia coli and characterization of TtMCO from the thermophilic bacterium Thermobaculum terrenum, the first laccase-like multi-copper oxidase (LMCO) from the distinct Phylum Chloroflexi. TtMCO has only 39% identity to its closest characterized homologue, CotA from Bacillus subtilis, but sequence and spectrophotometry confirmed copper coordination similar to that of LMCOs. TtMCO is extremely thermophilic with a half-time of inactivation of 2.24 days at 70°C and 350min at 80°C and pH 7, consistent with a hyperthermal habitat of the host. TtMCO was screened for activity against 56 chemically diverse substrates. It displayed limited activity on classical LMCO substrates, such as e.g. phenolics, transition metals, or bilirubin. Highest activities were observed for nitrogen-containing aromatic compounds, i.e. 1,8-diaminonaphtalene (Km=0.159mM, kcat=0.295s−1) and ABTS (Km=0.844mM, kcat=2.13s−1). The combined data suggest a distinct role of TtMCO and a substantial trade-off between activity and stability, compared to other characterized bacterial LMCOs, making it of interest in future protein engineering studies.

Complete genome sequence of 'Thermobaculum terrenum' type strain (YNP1).

‘Thermobaculum terrenum’ Botero et al. 2004 is the sole species within the proposed genus ‘Thermobaculum’. Strain YNP1T is the only cultivated member of an acid tolerant, extremely thermophilic species belonging to a phylogenetically isolated environmental clone group within the phylum Chloroflexi. At present, the name ‘Thermobaculum terrenum’ is not yet validly published as it contravenes Rule 30 (3a) of the Bacteriological Code. The bacterium was isolated from a slightly acidic extreme thermal soil in Yellowstone National Park, Wyoming (USA). Depending on its final taxonomic allocation, this is likely to be the third completed genome sequence of a member of the class Thermomicrobia and the seventh type strain genome from the phylum Chloroflexi. The 3,101,581 bp long genome with its 2,872 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

The phylogenetic placement of the non-phototrophic, Gram-positive thermophile ‘Thermobaculum terrenum’ and branching orders within the phylum ‘ Chloroflexi ’ inferred from gene order comparisons

The phylogenetic position of an anaerobic, non-spore-forming thermophile 'Thermobaculum terrenum' was investigated on the basis of gene order data from completely sequenced bacterial genomes. Gene order data can be an excellent source of phylogenetic information. Shared gene arrangements are unlikely to have arisen by chance convergence. They are likely to reflect common ancestry. 'Thermobaculum terrenum' was found to share three gene arrangements that are present uniquely in genomes of members of the phylum 'Chloroflexi', indicating convincingly that 'Thermobaculum terrenum' is a member of this phylum. Branching orders within the phylum 'Chloroflexi' were inferred by identifying monophyletic groups of species, which were circumscribed by characteristic gene arrangements. The branching orders thus inferred were in good agreement with previously reported phylogenies based on single 16S rRNA gene sequences and on multiple protein sequences. The gene order comparisons revealed a close phylogenetic affinity of 'Thermobaculum terrenum' to Sphaerobacter thermophilus and Thermomicrobium roseum.

Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia.

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).