No abstract

Isocitrate dehydrogenase (IDH) from the hyperthermophile Thermotoga maritima (TmIDH) catalyses NADP+‐ and metal‐dependent oxidative decarboxylation of isocitrate to α‐ketoglutarate. It belongs to the β‐decarboxylating dehydrogenase family and is the only hyperthermostable IDH identified within subfamily II. Furthermore, it is the only IDH that has been characterized as both dimeric and tetrameric in solution. We solved the crystal structure of the dimeric apo form of TmIDH at 2.2 Å. The R‐factor of the refined model was 18.5% (Rfree 22.4%). The conformation of the TmIDH structure was open and showed a domain rotation of 25–30° compared with closed IDHs. The separate domains were found to be homologous to those of the mesophilic mammalian IDHs of subfamily II and were subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that stabilization of the N‐ and C‐termini via long‐range electrostatic interactions were important for the higher thermostability of TmIDH. Moreover, the number of intra‐ and intersubunit ion pairs was higher and the ionic networks were larger compared with the mesophilic IDHs. Other factors likely to confer higher stability in TmIDH were a less hydrophobic and more charged accessible surface, a more hydrophobic subunit interface, more hydrogen bonds per residue and a few loop deletions. The residues responsible for the binding of isocitrate and NADP+ were found to be highly conserved between TmIDH and the mammalian IDHs and it is likely that the reaction mechanism is the same.

The gene encoding isocitrate dehydrogenase (IDH) of Methylococcus capsulatus (McIDH) was cloned and overexpressed in Escherichia coli. The purified enzyme was NAD+-dependent with a thermal optimum for activity at 55-60 degrees C and an apparent midpoint melting temperature (Tm) of 70 degrees C. Analytical ultracentrifugation (AUC) revealed a homotetrameric state, and McIDH thus represents the first homotetrameric NAD+-dependent IDH that has been characterized. Based on a structural alignment of McIDH and homotetrameric homoisocitrate dehydrogenase (HDH) from Thermus thermophilus (TtHDH), we identified the clasp-like domain of McIDH as a likely site for tetramerization. McIDH showed moreover, higher sequence identity (48%) to TtHDH than to previously characterized IDHs. Putative NAD+-IDHs with high sequence identity (48-57%) to McIDH were however identified in a variety of bacteria showing that NAD+-dependent IDHs are indeed widespread within the domain, Bacteria. Phylogenetic analysis including these new sequences revealed a close relationship with eukaryal allosterically regulated NAD+-IDH and the subfamily III of IDH was redefined to include bacterial NAD+- and NADP+-dependent IDHs. This apparent relationship suggests that the mitochondrial genes encoding NAD+-IDH are derived from the McIDH-like IDHs.

Deep-sea hydrothermal vents are amongst the most extreme environments on Earth and represent interesting targets for marine bioprospecting and biodiscovery. The microbial communities in hydrothermal vents are often dominated by chemolithoautotrophs utilizing simple chemical compounds, though the full extent of their heterotrophic abilities is still being explored. In the bioprocessing industry, where degradation of complex organic materials often is a major challenge, new microbial solutions are heavily needed. To meet these needs, we have developed novel in situ incubators and tested if deployment of recalcitrant materials from fish farming and wood-pulping industries introduced changes in the microbial community structure in hot marine hydrothermal sediments. The incubation chambers were deployed in sediments at the Bruse vent site located within the Jan Mayen vent field for 1 year, after which the microbial populations in the chambers were profiled by 16S rRNA Ion Torrent amplicon sequencing. A total of 921 operational taxonomic units (OTUs) were assigned into 74 different phyla where differences in community structure were observed depending on the incubated material, chamber depth below the sea floor and/or temperature. A high fraction of putative heterotrophic microbial lineages related to cultivated members within the Thermotogales were observed. However, considerable fractions of previously uncultivated and novel Thermotogales and Bacteroidetes were also identified. Moreover, several novel lineages (e.g., members within the DPANN superphylum, unidentified archaeal lineages, unclassified Thermoplasmatales and Candidatus division BRC-1 bacterium) of as-yet uncultivated thermophilic archaea and bacteria were identified. Overall, our data illustrate that amendment of hydrothermal vent communities by in situ incubation of biomass induces shifts in community structure toward increased fractions of heterotrophic microorganisms. The technologies utilized here could aid in subsequent metagenomics-based enzyme discovery for diverse industries.

The Virus-X—Viral Metagenomics for Innovation Value—project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.