1H, 15N and 13C backbone and side chain solution NMR assignments of the TPM domain-containing protein of the thermophilic bacterium Rhodothermus marinus.

The InterPro family IPR007621 TPM_phosphatase is a widely conserved family of protein domains found in prokaryotes, plants and invertebrates. Despite similar predicted protein folding, members of this family are involved in different cellular processes. In recent years, the structural and biochemical characterization of evolutionarily divergent TPM domains has shown their ability to hydrolyze phosphate groups of different substrates. However, there are still inaccurate functional annotations and uncertain relationships between the structure and function of this domain family. We here report the 1H, 13C, and 15N backbone and sidechain resonances of the TPM domain of a predicted TPM domain-containing protein of the thermophilic bacterium Rhodothermus marinus. These data will lay the groundwork for future NMR-based investigations, contributing to a thorough comprehension of the intricate aspects governing the interplay between structure and function of TPM domains. Additionally, they will unlock opportunities to explore dynamic structural changes, providing valuable insights into the molecular mechanisms underlying the evolutionary adaptations to extreme environmental conditions within this protein family.

A novel membraneless ß-glucan/O2 enzymatic fuel cell based on ß-glucosidase (RmBgl3B)/pyranose dehydrogenase (AmPDH) co-immobilized onto buckypaper electrode.

A novel membraneless ß-glucan/O2 enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus ß-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction. The fuel for the bioanode was produced by enzymatic hydrolysis of ß-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel. The maximum power density of the assembled ß-glucan/O2 biofuel cell reached 26.3 ±â€¯4.6 µWcm-2 at 0.36 V in phosphate buffer containing 0.5 % (w/v) ß-glucan at 40 °C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that ß-glucan can be employed as fuel in an enzymatic biofuel cell.

Effect of highly branched α-glucans synthesized by dual glycosyltransferases on the glucose release rate.

Increasing α-1,6 linkages in starch molecules generates a large amount of α-limit dextrins (α-LDx) during α-amylolysis, which decelerate the release of glucose at the intestinal α-glucosidase level. This study synthesized highly branched α-glucans from sucrose using Neisseria polysaccharea amylosucrase and Rhodothermus obamensis glycogen branching enzyme to enhance those of slowly digestible property. The synthesized α-glucans (Mw: 1.7-4.9 × 107 g mol-1) were mainly composed of α-1,4 linkages and large proportions of α-1,6 linkages (7.5%-9.9%). After treating the enzymatically synthesized α-glucans with the human α-amylase, the quantity of branched α-LDx (36.2%-46.7%) observed was higher than that for amylopectin (26.8%) and oyster glycogen (29.1%). When the synthetic α-glucans were hydrolyzed by mammalin α-glucosidases, the glucose generation rate decreased because the amount of embedded branched α-LDx increased. Therefore, the macro-sized branched α-glucans with high α-LDx has the potential to be used as slowly digestible material to attenuate postprandial glycemic response.

Rhodothermus bifroesti sp. nov., a thermophilic bacterium isolated from the basaltic subsurface of the volcanic island Surtsey.

Novel thermophilic heterotrophic bacteria were isolated from the subsurface of the volcanic island Surtsey off the south coast of Iceland. The strains were isolated from tephra core and borehole fluid samples collected below 70 m depth. The Gram-negative bacteria were rod-shaped (0.3-0.4 µm wide, 1.5-7 µm long), aerobic, non-sporulating and non-motile. Optimal growth was observed at 70 °C, at pH 7-7.5 and with 1% NaCl. Phylogenetic analysis identified the strains as members of the genus Rhodothermus. The type strain, ISCAR-7401T, was genetically distinct from its closest relatives Rhodothermus marinus DSM 4252T and Rhodothermus profundi PRI 2902T based on 16S rRNA gene sequence similarity (95.81 and 96.01%, respectively), genomic average nucleotide identity (73.73 and 72.61%, respectively) and digital DNA-DNA hybridization (17.6 and 16.9%, respectively). The major fatty acids of ISCAR-7401T were iso-C17:0, anteiso-C15:0, anteiso-C17:0 and iso-C15:0 (>10 %). The major isoprenoid quinone was MK-7 while phosphatidylethanolamine, diphosphatidylglycerol, an unidentified aminophospholipid and a phospholipid were the predominant polar lipid components. Based on comparative chemotaxonomic, genomic and phylogenetic analyses, we propose that the isolated strain represents a novel species of the genus Rhodothermus with the name Rhodothermus bifroesti sp. nov. The type strain is ISCAR-7401T (=DSM 112103T=CIP 111906T).

Genome-resolved metagenome and metatranscriptome analyses of thermophilic composting reveal key bacterial players and their metabolic interactions.

BACKGROUND: Composting is an important technique for environment-friendly degradation of organic material, and is a microbe-driven process. Previous metagenomic studies of composting have presented a general description of the taxonomic and functional diversity of its microbial populations, but they have lacked more specific information on the key organisms that are active during the process. RESULTS: Here we present and analyze 60 mostly high-quality metagenome-assembled genomes (MAGs) recovered from time-series samples of two thermophilic composting cells, of which 47 are potentially new bacterial species; 24 of those did not have any hits in two public MAG datasets at the 95% average nucleotide identity level. Analyses of gene content and expressed functions based on metatranscriptome data for one of the cells grouped the MAGs in three clusters along the 99-day composting process. By applying metabolic modeling methods, we were able to predict metabolic dependencies between MAGs. These models indicate the importance of coadjuvant bacteria that do not carry out lignocellulose degradation but may contribute to the management of reactive oxygen species and with enzymes that increase bioenergetic efficiency in composting, such as hydrogenases and N2O reductase. Strong metabolic dependencies predicted between MAGs revealed key interactions relying on exchange of H+, NH3, O2 and CO2, as well as glucose, glutamate, succinate, fumarate and others, highlighting the importance of functional stratification and syntrophic interactions during biomass conversion. Our model includes 22 out of 49 MAGs recovered from one composting cell data. Based on this model we highlight that Rhodothermus marinus, Thermobispora bispora and a novel Gammaproteobacterium are dominant players in chemolithotrophic metabolism and cross-feeding interactions. CONCLUSIONS: The results obtained expand our knowledge of the taxonomic and functional diversity of composting bacteria and provide a model of their dynamic metabolic interactions.

The Catalytic Mechanism of the Retaining Glycosyltransferase Mannosylglycerate Synthase.

To protect their intracellular proteins, extremophile microorganisms synthesize molecules called compatible solutes. These molecules are the result of the attachment of a small negatively charged molecule to a sugar molecule. It has been found that these molecules, not only protect the microorganism against osmotic stress but also against other extreme conditions. They can also confer protection against extreme conditions to isolated enzymes from different organisms making them an exciting prospect for potential biotechnological applications. One of the most widespread compatible solute in hyperthermophile organisms is the molecule 2-O-α-D-mannosyl-D-glycerate (MG). In addition to confer protection to proteins against extreme conditions, MG was found to prevent Alzheimer's ß-amyloid aggregation and reduce α-synuclein fibril formation in Parkinson's disease. In this work we studied, using computational methods, the catalytic mechanism of the synthesis of MG by the enzyme mannosylglycerate synthase (MGS) from the thermophilic bacteria Rhodothermus marinus.

Flexible Loop in Carbohydrate-Binding Module 48 Allosterically Modulates Substrate Binding of the 1,4-α-Glucan Branching Enzyme.

The 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) catalyzes the formation of α-1,6 branching points in starch and plays a key role in synthesis. To obtain mechanistic insights into the catalytic action of the enzyme, we first determined the crystal structure of GBE from Rhodothermus obamensis STB05 (RoGBE) to a resolution of 2.39 Å (PDB ID: 6JOY). The structure consists of three domains: domain A, domain C, and the carbohydrate-binding module 48 (CBM48). An engineered truncated mutant lacking the CBM48 domain (ΔCBM48) showed significantly reduced ligand binding affinity and enzyme activity. Comparison of the structures of RoGBE with other GBEs showed that CBM48 of RoGBE had a longer flexible loop. Truncation of the flexible loops resulted in reduced binding affinity and activity, thereby substantiating the importance of the optimum loop structure for catalysis. In essence, our study shows that CBM48, especially the flexible loop, plays an important role in substrate binding and enzymatic activity of RoGBE. Further, based on the structural analysis, kinetics, and activity assays on wild type and mutants, as well as homology modeling, we proposed a mechanistic model (called the "lid model") to illustrate how the flexible loop triggers substrate binding, ultimately leading to catalysis.

Purification and Amplification of DNA from Cellulolytic Bacteria: Application for Biogas Production from Crop Residues.

Polymerase chain reaction (PCR) is a popular molecular tool for detection of bacteria. PCR allows millions of copies of a target segment of DNA to be produced. The DNA is extracted from overnight grown cultures of pure bacterial isolates using either the organo-solvent method or a commercial DNA extraction kit. The quality and purity of the DNA is determined by performing gel electrophoresis on 0.8% agarose gel. The DNA is amplified by performing PCR assay. Bands of approximately 1.5 kb in size are obtained from the amplified products of DNA. The PCR products run on 1.5% agarose gel are visualized with UV light and imaged by gel documentation system. This chapter outlines the protocol for isolation and amplification of DNA from cellulolytic bacteria. Cellulolytic bacteria are considered a potential source of cellulases for pretreatment of crop residues during biogas production. PCR is considered a very powerful, sensitive, specific, fast, and reliable tool in molecular detection and diagnostics.

Two 1,4-α-glucan branching enzymes successively rearrange glycosidic bonds: A novel synergistic approach for reducing starch digestibility.

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.

Structural Flexibility of Peripheral Loops and Extended C-terminal Domain of Short Length Substrate Binding Protein from Rhodothermus marinus.

Substrate binding proteins (SBPs) bind to specific ligands in the periplasmic regions of cells and then bind to membrane proteins to participate in transport or signal transduction. Typically, SBPs consist of two α/ß domains and recognize the substrate by a flexible hinge region between the two domains. Conversely, the short-length SBPs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes. We previously determined the crystal structure of Rhodothermus marinus SBP (named as RmSBP), consisting of a single α/ß domain; however, the substrate recognition mechanism is still unclear. To better understand the functions of short length RmSBP, we performed a comprehensive study, involving comparative structure analysis, computational substrate docking, and X-ray crystallographic data. RmSBP shares a high level of similarity in the α/ß domain region with other SBPs, but it has a distinct topology in the C-terminal domain. The substrate binding model suggested that conformational changes in the peripheral region of RmSBP was required to recognize the substrate. We determined the crystal structures of RmSBP at pH 5.5, 6.0, and 7.5. RmSBP showed structural flexibility in the ß1-α2 loop, ß5-ß6 loop, and extended C-terminal domains, based on the electron density map and temperature B-factor analysis. These results provide information that will further our understanding on the functions of the short length SBP.

Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253.

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: ß-N-acetyl-glucosaminidases, ß-1,4-glucosidases/ß-xylosidases and macrolide ß-glucosidases. The RmNag3 with additional ß-lactamase domain clustered with the deepest rooted GH3-lineage of ß-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed ß-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide ß-glucosidases from Actinomycetes. The ß-xylosidases, RmXyl3A and RmXyl3B, and the ß-glucosidases RmBgl3A and RmBgl3C clustered within the major ß-glucosidases/ß-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed ß-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed ß-1,4-glucosidase/ß-xylosidase activity while RmBgl3C was active on pNP-ß-Glc and ß-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization.

Cultivation technology development of Rhodothermus marinus DSM 16675.

This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.

Heterologous expression, purification and biochemical characterization of a new endo-1,4-ß-xylanase from Rhodothermaceae bacterium RA.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2-65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Expression and characterization of an extremely thermophilic 1,4-α-glucan branching enzyme from Rhodothermus obamensis STB05.

A gene encoding 1,4-α-glucan branching enzyme (GBE, EC 2.4.1.18) from the extremely thermophilic bacterium Rhodothermus obamensis STB05 was successfully cloned and expressed in Escherichia coli. Extracellular expression of the recombinant enzyme (R.o-GBE) was achieved with a yield of 1080 mg/L. Then it was purified and further characterized biochemically. R.o-GBE was optimally active at pH 7.0 and 65 °C. It remained stable at temperatures up to 80 °C and had a half-life at 85 °C of approximately 31 min. Far-UV circular dichroism and intrinsic fluorescence analyses revealed that high temperatures reduced its activity by changing the secondary and tertiary structure of R.o-GBE. The enzyme had broad pH stability between pH 3.0 and 11.0 at 4 °C, and preferred weakly acidic conditions at high temperatures. None of the metal ions enhanced the activity of R.o-GBE, but Ca2+ may be required for its activity. Its specific activity with amylopectin was 6651 U/mg, which is much higher than that reported for other GBEs. Its excellent thermostability, broad pH stability, and high specific activity make R.o-GBE highly suitable for industrial applications.

Enantiodivergent α-Amino C-H Fluoroalkylation Catalyzed by Engineered Cytochrome P450s.

The introduction of fluoroalkyl groups into organic compounds can significantly alter pharmacological characteristics. One enabling but underexplored approach for the installation of fluoroalkyl groups is selective C( sp3)-H functionalization due to the ubiquity of C-H bonds in organic molecules. We have engineered heme enzymes that can insert fluoroalkyl carbene intermediates into α-amino C( sp3)-H bonds and enable enantiodivergent synthesis of fluoroalkyl-containing molecules. Using directed evolution, we engineered cytochrome P450 enzymes to catalyze this abiological reaction under mild conditions with total turnovers (TTN) up to 4070 and enantiomeric excess (ee) up to 99%. The iron-heme catalyst is fully genetically encoded and configurable by directed evolution so that just a few mutations to the enzyme completely inverted product enantioselectivity. These catalysts provide a powerful method for synthesis of chiral organofluorine molecules that is currently not possible with small-molecule catalysts.

Non-classical secretion of 1,4-alpha-glucan branching enzymes without signal peptides in Escherichia coli.

1,4-α-Glucan branching enzyme (GBE, EC. 2.4.1.18), which plays a key role in the synthesis of starch and glycogen, has been overexpressed in E. coli as an intracellular enzyme by many researchers. In this study, it was found that the GBEs from Geobacillus thermoglucosidans and Rhodothermus obamensis were secreted into the culture medium when they were expressed separately, in E. coli. This occurred despite the absence of any signal peptide. In fact, although bioinformatics tools predicted that both of these proteins would localize to the cytoplasm, a high level of expression and non-classical secretion was found to achieve without addition of the inducer isopropyl ß-d-thiogalactopyranoside. Further experiments revealed that secretion was a two-step process that occurred via the periplasmic space. Results excluded the involvement of the Sec pathway or the TAT pathway. Instead, the findings indicated a relationship between cell membrane integrity and the secretion of the two GBEs, and suggested that their N-termini play an essential role in their expression and secretion.

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide-Dependent Methanol Dehydrogenase Activity.

Lanthanide (Ln)-dependent methanol dehydrogenases (MDHs) have recently been shown to be widespread in methylotrophic bacteria. Along with the core MDH protein, XoxF, these systems contain two other proteins, XoxG (a c-type cytochrome) and XoxJ (a periplasmic binding protein of unknown function), about which little is known. In this work, we have biochemically and structurally characterized these proteins from the methyltroph Methylobacterium extorquens AM1. In contrast to results obtained in an artificial assay system, assays of XoxFs metallated with LaIII , CeIII , and NdIII using their physiological electron acceptor, XoxG, display Ln-independent activities, but the Km for XoxG markedly increases from La to Nd. This result suggests that XoxG's redox properties are tuned specifically for lighter Lns in XoxF, an interpretation supported by the unusually low reduction potential of XoxG (+172 mV). The X-ray crystal structure of XoxG provides a structural basis for this reduction potential and insight into the XoxG-XoxF interaction. Finally, the X-ray crystal structure of XoxJ reveals a large hydrophobic cleft and suggests a role in the activation of XoxF. These studies enrich our understanding of the underlying chemical principles that enable the activity of XoxF with multiple lanthanides in vitro and in vivo.

Machine learning-assisted directed protein evolution with combinatorial libraries.

To reduce experimental effort associated with directed protein evolution and to explore the sequence space encoded by mutating multiple positions simultaneously, we incorporate machine learning into the directed evolution workflow. Combinatorial sequence space can be quite expensive to sample experimentally, but machine-learning models trained on tested variants provide a fast method for testing sequence space computationally. We validated this approach on a large published empirical fitness landscape for human GB1 binding protein, demonstrating that machine learning-guided directed evolution finds variants with higher fitness than those found by other directed evolution approaches. We then provide an example application in evolving an enzyme to produce each of the two possible product enantiomers (i.e., stereodivergence) of a new-to-nature carbene Si-H insertion reaction. The approach predicted libraries enriched in functional enzymes and fixed seven mutations in two rounds of evolution to identify variants for selective catalysis with 93% and 79% ee (enantiomeric excess). By greatly increasing throughput with in silico modeling, machine learning enhances the quality and diversity of sequence solutions for a protein engineering problem.

Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by an Engineered Hemoprotein.

Chiral 1,2-amino alcohols are widely represented in biologically active compounds from neurotransmitters to antivirals. While many synthetic methods have been developed for accessing amino alcohols, the direct aminohydroxylation of alkenes to unprotected, enantioenriched amino alcohols remains a challenge. Using directed evolution, we have engineered a hemoprotein biocatalyst based on a thermostable cytochrome c that directly transforms alkenes to amino alcohols with high enantioselectivity (up to 2500 TTN and 90 % ee) under anaerobic conditions with O-pivaloylhydroxylamine as an aminating reagent. The reaction is proposed to proceed via a reactive iron-nitrogen species generated in the enzyme active site, enabling tuning of the catalyst's activity and selectivity by protein engineering.

Converting Galactose into the Rare Sugar Talose with Cellobiose 2-Epimerase as Biocatalyst.

Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of ß-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of d-talose from d-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed. First, a mutant library of RmCE was created to try and increase the activity on monosaccharide substrates. Next, two residues from RmCE were introduced in the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) (S99M/Q371F), increasing the kcat twofold.