Comparative studies on substrate specificity of succinic semialdehyde reductase from Gluconobacter oxydans and glyoxylate reductase from Acetobacter aceti.

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the ß-HAD superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate-specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes.

Computational Analysis of Activation of Dimerized Epidermal Growth Factor Receptor Kinase Using the String Method and Markov State Model.

Epidermal growth factor receptor (EGFR) activation is accompanied by dimerization. During the activation of the intracellular kinase domain, two EGFR kinases form an asymmetric dimer, and one side of the dimer (receiver) is activated. Using the string method and Markov state model (MSM), we performed a computational analysis of the structural changes in the activation of the EGFR dimer in this study. The string method reveals the minimum free-energy pathway (MFEP) from the inactive to active structure. The MSM was constructed from numerous trajectories of molecular dynamics simulations around the MFEP, which revealed the free-energy map of structural changes. In the activation of the receiver kinase, the unfolding of the activation loop (A-loop) is followed by the rearrangement of the C-helix, as observed in other kinases. However, unlike other kinases, the free-energy map of EGFR at the asymmetric dimer showed that the active state yielded the highest stability and revealed how interactions at the dimer interface induced receiver activation. As the H-helix of the activator approaches the C-helix of the receiver during activation, the A-loop unfolds. Subsequently, L782 of the receiver enters the pocket between the G- and H-helices of the activator, leading to a rearrangement of the hydrophobic residues around L782 of the receiver, which constitutes a structural rearrangement of the C-helix of the receiver from an outward to an inner position. The MSM analysis revealed long-time scale trajectories via kinetic Monte Carlo.

Draft genome of Parageobacillus thermoglucosidasius, a member of hydrogenogenic carbon monoxide utilizers, isolated from a freshwater lake sediment.

Parageobacillus thermoglucosidasius is a facultatively anaerobic thermophile and possesses carbon monoxide dehydrogenase and hydrogenase for carbon monoxide (CO) oxidation and hydrogen production, respectively. In this study, we report a draft genome of P. thermoglucosidasius isolated from a freshwater sediment, expanding our knowledge on the distribution of CO utilizers.

Comparison of the Molecular Motility of Tubulin Dimeric Isoforms: Molecular Dynamics Simulations and Diffracted X-ray Tracking Study.

Tubulin has been recently reported to form a large family consisting of various gene isoforms; however, the differences in the molecular features of tubulin dimers composed of a combination of these isoforms remain unknown. Therefore, we attempted to elucidate the physical differences in the molecular motility of these tubulin dimers using the method of measurable pico-meter-scale molecular motility, diffracted X-ray tracking (DXT) analysis, regarding characteristic tubulin dimers, including neuronal TUBB3 and ubiquitous TUBB5. We first conducted a DXT analysis of neuronal (TUBB3-TUBA1A) and ubiquitous (TUBB5-TUBA1B) tubulin dimers and found that the molecular motility around the vertical axis of the neuronal tubulin dimer was lower than that of the ubiquitous tubulin dimer. The results of molecular dynamics (MD) simulation suggest that the difference in motility between the neuronal and ubiquitous tubulin dimers was probably caused by a change in the major contact of Gln245 in the T7 loop of TUBB from Glu11 in TUBA to Val353 in TUBB. The present study is the first report of a novel phenomenon in which the pico-meter-scale molecular motility between neuronal and ubiquitous tubulin dimers is different.

Construction of multiple metagenome assembled genomes containing carbon monoxide dehydrogenases from anaerobic carbon monoxide enrichment cultures.

Despite its toxicity to many organisms, including most prokaryotes, carbon monoxide (CO) is utilized by some aerobic and anaerobic prokaryotes. Hydrogenogenic CO utilizers employ carbon monoxide dehydrogenase (CODH) and energy-converting hydrogenase (ECH) to oxidize CO and reduce protons to produce H2. Those prokaryotes constitute a rare biosphere and are difficult to detect even with PCR amplification and with metagenomic analyses. In this study, anaerobic CO-enrichment cultures followed by construction of metagenome assembled genomes (MAGs) detected high-quality MAGs from potential hydrogenogenic CO utilizers. Of 32 MAGs constructed, 5 were potential CO utilizer harboring CODH genes. Of the five MAGs, two were classified into the genus Thermolithobacter on the basis of 16S rRNA sequence identity, related to Carboxydocella tharmautotrophica 41, with an average nucleotide identity (ANI) of approximately 72%. Additionally, two were related to Geoglobus acetivorans with ANI values ranging from 75 to 77% to G. acetivorans SBH6, and one MAG was identified as Desulfotomaculum kuznetsovii with an ANI > 96% to D. kuznetsovii DSM 6115. The two Thermolithobacter MAGs identified in this study contained CODH-ECH gene clusters, and were therefore identified as potential hydrogenogenic CO utilizers. However, these MAGs harbored three CODH gene clusters that showed distinct physiological functions in addition to CODH-ECH gene clusters. In total, the five potential CO utilizer MAGs contained sixteen CODH genes. Among those CODHs, four sets did not cluster with any known CODH protein sequences (with an identity of > 90%), and the CODH database was expanded.

Isolation, Genomic Sequence and Physiological Characterization of Parageobacillus sp. G301, an Isolate Capable of Both Hydrogenogenic and Aerobic Carbon Monoxide Oxidation.

Prokaryotes that can oxidize carbon monoxide (CO oxidizers) can use this gas as a source of carbon or energy. They oxidize carbon monoxide with carbon monoxide dehydrogenases (CODHs): these are divided into nickel-containing CODH (Ni-CODH), which are sensitive to O2, and molybdenum-containing CODH (Mo-CODH), which can function aerobically. The oxygen conditions required for CO oxidizers to oxidize CO may be limited, as those which have been isolated and characterized so far contain either Ni- or Mo-CODH. Here, we report a novel CO oxidizer, Parageobacillus sp. G301, which is capable of CO oxidation using both types of CODH based on genomic and physiological characterization. This thermophilic, facultatively anaerobic Bacillota bacterium was isolated from the sediments of a freshwater lake. Genomic analyses revealed that strain G301 possessed both Ni-CODH and Mo-CODH. Genome-based reconstruction of its respiratory machinery and physiological investigations indicated that CO oxidation by Ni-CODH was coupled with H2 production (proton reduction), whereas CO oxidation by Mo-CODH was coupled with O2 reduction under aerobic conditions and nitrate reduction under anaerobic conditions. G301 would thus be able to thrive via CO oxidation under a wide range of conditions, from aerobic environments to anaerobic environments, even with no terminal electron acceptors other than protons. Comparative genome analyses revealed no significant differences in genome structures and encoded cellular functions, except for CO oxidation between CO oxidizers and non-CO oxidizers in the genus Parageobacillus; CO oxidation genes are retained exclusively for CO metabolism and related respiration. IMPORTANCE Microbial CO oxidation has received much attention because it contributes to global carbon cycling in addition to functioning as a remover of CO, which is toxic to many organisms. Some microbial CO oxidizers, including both bacteria and archaea, exhibit sister relationships with non-CO oxidizers even in genus-level monophyletic groups. In this study, we demonstrated that a new isolate, Parageobacillus sp. G301, is capable of both anaerobic (hydrogenogenic) and aerobic CO oxidation, which has not been previously reported. The discovery of this new isolate, which is versatile in CO metabolism, will accelerate research on CO oxidizers with diverse CO metabolisms, expanding our understanding of microbial diversity. Through comparative genomic analyses, we propose that CO oxidation genes are not essential genetic elements in the genus Parageobacillus, providing insights into the factors which shape the punctate distribution of CO oxidizers in the prokaryote tree, even in genus-level monophyletic groups.

Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction.

MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.

Biome-specific distribution of Ni-containing carbon monoxide dehydrogenases.

Ni-containing carbon monoxide dehydrogenase (Ni-CODH) plays an important role in the CO/CO2-based carbon and energy metabolism of microbiomes. Ni-CODH is classified into distinct phylogenetic clades, A-G, with possibly distinct cellular roles. However, the types of Ni-CODH clade used by organisms in different microbiomes are unknown. Here, we conducted a metagenomic survey of a protein database to determine the relationship between the phylogeny and biome distribution of Ni-CODHs. Clustering and phylogenetic analyses showed that the metagenome assembly-derived Ni-CODH sequences were distributed in ~ 60% Ni-CODH clusters and in all Ni-CODH clades. We also identified a novel Ni-CODH clade, clade H. Biome mapping on the Ni-CODH phylogenetic tree revealed that Ni-CODHs of almost all the clades were found in natural aquatic environmental and engineered samples, whereas those of specific subclades were found only in host-associated samples. These results are comparable with our finding that the diversity in the phylum-level taxonomy of host-associated Ni-CODH owners is statistically different from those of the other biomes. Our findings suggest that while Ni-CODH is a ubiquitous enzyme produced across diverse microbiomes, its distribution in each clade is biased and mainly affected by the distinct composition of microbiomes.

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA-selenium complexes.

Complete Genome Sequence of Pseudomonas stutzeri Strain F2a, Isolated from Seleniferous Soil.

Pseudomonas stutzeri is a potential candidate for bioremediation of selenium-contaminated grounds and waters. Here, we report the complete genome sequence of a novel strain, F2a, which was isolated from a seleniferous area of Punjab, India. The genome sequence provides insight into the potential selenium oxyanion-reducing activity of this strain.

Diversity analysis of thermophilic hydrogenogenic carboxydotrophs by carbon monoxide dehydrogenase amplicon sequencing using new primers.

The microbial H2-producing (hydrogenogenic) carbon monoxide (CO)-oxidizing activity by the membrane-associated CO dehydrogenase (CODH)/energy-converting hydrogenase (ECH) complex is an important metabolic process in the microbial community. However, the studies on hydrogenogenic carboxydotrophs had to rely on inherently cultivation and isolation methods due to their rare abundance, which was a bottleneck in ecological study. Here, we provided gene-targeted sequencing method for the diversity estimation of thermophilic hydrogenogenic carboxydotrophs. We designed six new degenerate primer pairs which effectively amplified the coding regions of CODH genes forming gene clusters with ECH genes (CODHech genes) in Firmicutes which includes major thermophilic hydrogenogenic carboxydotrophs in terrestrial thermal habitats. Amplicon sequencing by these primers using DNAs from terrestrial hydrothermal sediments and CO-gas-incubated samples specifically detected multiple CODH genes which were identical or phylogenetically related to the CODHech genes in Firmictes. Furthermore, we found that phylogenetically distinct CODHech genes were enriched in CO-gas-incubated samples, suggesting that our primers detected uncultured hydrogenogenic carboxydotrophs as well. The new CODH-targeted primers provided us with a fine-grained (~ 97.9% in nucleotide sequence identity) diversity analysis of thermophilic hydrogenogenic carboxydotrophs by amplicon sequencing and will bolster the ecological study of these microorganisms.

Structural changes induced by ligand binding drastically increase the thermostability of the Ser/Thr protein kinase TpkD from Thermus thermophilus HB8.

Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand-induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP-binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins.

Genetic Engineering of Carbon Monoxide-dependent Hydrogen-producing Machinery in Parageobacillus thermoglucosidasius.

The metabolic engineering of carbon monoxide (CO) oxidizers has the potential to create efficient biocatalysts to produce hydrogen and other valuable chemicals. We herein applied markerless gene deletion to CO dehydrogenase/energy-converting hydrogenase (CODH/ECH) in the thermophilic facultative anaerobe, Parageobacillus thermoglucosidasius. We initially compared the transformation efficiency of two strains, NBRC 107763T and TG4. We then disrupted CODH, ECH, and both enzymes in NBRC 107763T. The characterization of growth in all three disruptants under 100% CO demonstrated that both enzymes were essential for CO-dependent growth with hydrogen production in P. thermoglucosidasius. The present results will become a platform for the further metabolic engineering of this organism.

Draft Genome Sequence of Thermanaeromonas sp. Strain C210, Isolated in the Presence of Carbon Monoxide.

The genus Thermanaeromonas comprises two species of thermophilic, strictly anaerobic, spore-forming bacteria. Here, we report the draft genome sequence of Thermanaeromonas sp. strain C210, which was first isolated in the presence of carbon monoxide. The genome sequence provides insight into carbon monoxide-dependent metabolism for members of the genus Thermanaeromonas.

Carbon monoxide-dependent transcriptional changes in a thermophilic, carbon monoxide-utilizing, hydrogen-evolving bacterium Calderihabitans maritimus KKC1 revealed by transcriptomic analysis.

Calderihabitans maritimus KKC1 is a thermophilic, carbon monoxide (CO)-utilizing, hydrogen-evolving bacterium that harbors seven cooS genes for anaerobic CO dehydrogenases and six hyd genes for [NiFe] hydrogenases and capable of using a variety of electron acceptors coupled to CO oxidation. To understand the relationships among these unique features and the transcriptional adaptation of the organism to CO, we performed a transcriptome analysis of C. maritimus KKC1 grown under 100% CO and N2 conditions. Of its 3114 genes, 58 and 32 genes were significantly upregulated and downregulated in the presence of CO, respectively. A cooS-ech gene cluster, an "orphan" cooS gene, and bidirectional hyd genes were upregulated under CO, whereas hydrogen-uptake hyd genes were downregulated. Transcriptional changes in anaerobic respiratory genes supported the broad usage of electron acceptors in C. maritimus KKC1 under CO metabolism. Overall, the majority of the differentially expressed genes were oxidoreductase-like genes, suggesting metabolic adaptation to the cellular redox change upon CO oxidation. Moreover, our results suggest a transcriptional response mechanism to CO that involves multiple transcription factors, as well as a CO-responsive transcriptional activator (CooA). Our findings shed light on the diverse mechanisms for transcriptional and metabolic adaptations to CO in CO-utilizing and hydrogen-evolving bacteria.

Anaerobic and hydrogenogenic carbon monoxide-oxidizing prokaryotes: Versatile microbial conversion of a toxic gas into an available energy.

Carbon monoxide (CO) is a gas that is toxic to various organisms including humans and even microbes; however, it has low redox potential, which can fuel certain microbes, namely, CO oxidizers. Hydrogenogenic CO oxidizers utilize an energy conservation system via a CO dehydrogenase/energy-converting hydrogenase complex to produce hydrogen gas, a zero emission fuel, by CO oxidation coupled with proton reduction. Biochemical and molecular biological studies using a few model organisms have revealed their enzymatic reactions and transcriptional response mechanisms using CO. Biotechnological studies for CO-dependent hydrogen production have also been carried out with these model organisms. In this chapter, we review recent advances in the studies of these microbes, which reveal their unique and versatile metabolic profiles and provides future perspectives on ecological roles and biotechnological applications. Over the past decade, the number of isolates has doubled (37 isolates in 5 phyla, 20 genera, and 32 species). Some of the recently isolated ones show broad specificity to electron acceptors. Moreover, accumulating genomic information predicts their unique physiologies and reveals their phylogenomic relationships with novel potential hydrogenogenic CO oxidizers. Combined with genomic database surveys, a molecular ecological study has unveiled the wide distribution and low abundance of these microbes. Finally, recent biotechnological applications of hydrogenogenic CO oxidizers have been achieved via diverse approaches (e.g., metabolic engineering and co-cultivation), and the identification of thermophilic facultative anaerobic CO oxidizers will promote industrial applications as oxygen-tolerant biocatalysts for efficient hydrogen production by genomic engineering.

Hydration properties of a protein at low and high pressures: Physics of pressure denaturation.

Using experimentally determined structures of ubiquitin at 1 and 3000 bar, we generate sufficiently large ensembles of model structures in the native and pressure-induced (denatured) states by means of molecular dynamics simulations with explicit water. We calculate the values of a free-energy function (FEF), which comprises the hydration free energy (HFE) and the intramolecular (conformational) energy and entropy, for the two states at 1 and 3000 bar. The HFE and the conformational entropy, respectively, are calculated using our statistical-mechanical method, which has recently been shown to be accurate, and the Boltzmann-quasi-harmonic method. The HFE is decomposed into a variety of physically insightful components. We show that the FEF of the native state is lower than that of the denatured state at 1 bar, whereas the opposite is true at 3000 bar, thus being successful in reproducing the pressure denaturation. We argue that the following two quantities of hydration play essential roles in the denaturation: the WASA-dependent term in the water-entropy loss upon cavity creation for accommodating the protein (WASA is the water-accessible surface area of the cavity) and the protein-water Lennard-Jones interaction energy. At a high pressure, the mitigation of the serious water crowding in the system is the most important, and the WASA needs to be sufficiently enlarged with the increase in the excluded-volume being kept as small as possible. The denatured structure thus induced is characterized by the water penetration into the protein interior. The pressure denaturation is accompanied by a significantly large gain of water entropy.

Draft Genome Sequences of Two Thermophilic Moorella sp. Strains, Isolated from an Acidic Hot Spring in Japan.

The thermophilic Moorella sp. strains E308F and E306M were isolated from an acidic hot spring in Japan. Here, we report the draft genome sequences of E308F (3.06 Mbp; G+C content, 54.0%) and E306M (2.99 Mbp; G+C content, 54.4%), to advance the genomic information available on the genus Moorella.

An accurate and rapid method for calculating hydration free energies of a variety of solutes including proteins.

A new method is developed for calculating hydration free energies (HFEs) of polyatomic solutes. The solute insertion is decomposed into the creation of a cavity in water matching the geometric characteristics of the solute at the atomic level (process 1) and the incorporation of solute-water van der Waals and electrostatic interactions (process 2). The angle-dependent integral equation theory combined with our morphometric approach and the three-dimensional interaction site model theory are applied to processes 1 and 2, respectively. Neither a stage of training nor parameterization is necessitated. For solutes with various sizes including proteins, the HFEs calculated by the new method are compared to those obtained using a molecular dynamics simulation based on solution theory in energy representation (the ER method developed by Matubayasi and co-workers), currently the most reliable tool. The agreement is very good especially for proteins. The new method is characterized by the following: The calculation can rapidly be finished; a solute possessing a significantly large total charge can be handled without difficulty; and since it yields not only the HFE but also its many physically insightful energetic and entropic components, it is best suited to the elucidation of mechanisms of diverse phenomena such as the receptor-ligand binding, different types of molecular recognition, and protein folding, denaturation, and association.

Draft Genome Sequence of Parageobacillus thermoglucosidasius Strain TG4, a Hydrogenogenic Carboxydotrophic Bacterium Isolated from a Marine Sediment.

Parageobacillus thermoglucosidasius possesses biotechnological potential for fuel generation. Here, we report the draft genome sequence of P. thermoglucosidasius strain TG4, which was first isolated from a marine sediment. The genome sequence provides insight into the plasmid diversity and carbon monoxide-dependent hydrogen production capacity of P. thermoglucosidasius.