Copper homeostasis is a fundamental process in organisms, characterised by unique pathways that have evolved to meet specific needs while preserving core resistance mechanisms. While these systems are well-documented in model bacteria, information on copper resistance in species adapted to cold environments is scarce. This study investigates the potential genes related to copper homeostasis in the genome of Bizionia argentinensis (JUB59-T), a psychrotolerant bacterium isolated from Antarctic seawater. We identified several genes encoding proteins analogous to those crucial for copper homeostasis, including three sequences of copper-transport P1B-type ATPases. One of these, referred to as BaCopA1, was chosen for cloning and expression in Saccharomyces cerevisiae. BaCopA1 was successfully integrated into yeast membranes and subsequently extracted with detergent. The purified BaCopA1 demonstrated the ability to catalyse ATP hydrolysis at low temperatures. Structural models of various BaCopA1 conformations were generated and compared with mesophilic and thermophilic homologous structures. The significant conservation of critical residues and structural similarity among these proteins suggest a shared reaction mechanism for copper transport. This study is the first to report a psychrotolerant P1B-ATPase that has been expressed and purified in a functional form.

Archaeal viruses are one of the most mysterious parts of the virosphere because of their diverse morphologies and unique genome contents. The crenarchaeal viruses are commonly found in high temperature and acidic hot springs, and the number of identified crenarchaeal viruses is being rapidly increased in recent two decades. Over fifty viruses infecting the members of the order Sulfolobales have been identified, most of which are from hot springs distributed in the United States, Russia, Iceland, Japan, and Italy. To further expand the reservoir of viruses infecting strains of Sulfolobaceae, we investigated virus diversity through cultivation-dependent approaches in hot springs in Tengchong, Yunnan, China. Eight different virus-like particles were detected in enrichment cultures, among which five new archaeal viruses were isolated and characterized. We showed that these viruses can infect acidophilic hyperthermophiles belonging to three different genera of the family Sulfolobaceae, namely, Saccharolobus, Sulfolobus, and Metallosphaera. We also compared the lipid compositions of the viral and cellular membranes and found that the lipid composition of some viral envelopes was very different from that of the host membrane. Collectively, our results showed that the Tengchong hot springs harbor highly diverse viruses, providing excellent models for archaeal virus-host studies.

A novel thermoacidophilic archeaon, strain J1T (=DSM 112778T,=JCM 34702T), was isolated from a hot pool in a volcanic area of Java, Indonesia. Cells of the strain were irregular, motile cocci of 1.0-1.2 µm diameter. Aerobic, organoheterotrophic growth with casamino acids was observed at an optimum temperature of 70 °C in a range of 55-78 °C and at an optimum pH of 3 in a range of 1.5 to 5. Various organic compounds were utilized, including a greater variety of sugars than has been reported for growth of other species of the genus. Chemolithoautotrophic growth was observed with reduced sulphur compounds, including mineral sulphides. Ferric iron was reduced during anaerobic growth with elemental sulphur. Cellular lipids were calditoglycerocaldarchaeol and caldarchaeol with some derivates. The organism contained the respiratory quinone caldariellaquinone. On the basis of phylogenetic and chemotaxonomic comparison with its closest relatives, it was concluded that strain J1T represents a novel species, for which the name Metallosphaera javensis is proposed. Low DNA-DNA relatedness values (16S rRNA gene <98.4%, average nucleotide identity (ANI) <80.1%) distinguished J1T from other species of the genus Metallosphaera and the DNA G+C content of 47.3% is the highest among the known species of the genus.

A novel hyperthermophilic, acidophilic and facultatively anaerobic archaeon, strain KN-1T, was isolated from Unzen hot spring in Japan and characterized. The cells of KN-1T were irregular cocci with a diameter of 1.0-3.0 µm that grew at 55-87.5 °C (optimum: 75 °C) and pH 1.0-5.5 (optimum: 3.0). Chemolithoautotrophic growth of KN-1T occurred in the presence of S0 or H2 under oxic conditions. Under anoxic conditions, KN-1T grew with S0, ferric citrate and FeCl3 as electron acceptors. A phylogenetic analysis of 16S rRNA gene sequences showed that the species most closely related to KN-1T was Stygiolobus azoricus JCM 9 021T, with 98.9 % sequence identity, indicating that strain KN-1T belongs to the genus Stygiolobus. This genus has been considered to consist of obligate anaerobes since its description in 1991. However, KN-1T grew under oxic, microoxic and anoxic conditions. Moreover, KN-1Tutilized various complex substrates and some sugars as carbon or energy sources, which is also different from S. azoricus JCM 9 021T. The average nucleotide identity and amino acid identity values between KN-1T and S. azoricus JCM 9 021T were 79.4 and 76.1 %, respectively, indicating that KN-1T represents a novel species. Its main polar lipids were calditoglycerocaldarchaeol and caldarchaeol, and its DNA G+C content was 40.1 mol%. We also found that S. azoricus JCM 9021T grew under microoxic conditions in the presence of H2 as an electron donor, indicating that this genus does not comprise obligate anaerobes. Based on this polyphasic taxonomic analysis, we propose the novel species, Stygiolobus caldivivus sp. nov., whose type strain is KN-1T (=JCM 34 622T=KCTC 4 293T).

When overexpressed as an immature enzyme in the mesophilic bacterium Escherichia coli, recombinant homoserine dehydrogenase from the hyperthermophilic archaeon Sulfurisphaera tokodaii (StHSD) was markedly activated by heat treatment. Both the apo- and holo-forms of the immature enzyme were successively crystallized, and the two structures were determined. Comparison among the structures of the immature enzyme and previously reported structures of mature enzymes revealed that a conformational change in a flexible part (residues 160-190) of the enzyme, which encloses substrates within the substrate-binding pocket, is smaller in the immature enzyme. The immature enzyme, but not the mature enzyme, formed a complex that included NADP+, despite its absence during crystallization. This indicates that the opening to the substrate-binding pocket in the immature enzyme is not sufficient for substrate-binding, efficient catalytic turnover or release of NADP+. Thus, specific conformational changes within the catalytic region appear to be responsible for heat-induced activation.

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5-8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme\'s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.

O6-Methylguanine-DNA methyltransferase (MGMT) removes cytotoxic O6-alkyl adducts on the guanine base and protects the cell from genomic damage induced by alkylating agents. Although there are reports of computational studies on the activity of the enzyme with mutations at tyrosine residues, no studies concerning the crystal structure of its mutants have been found. In this study, the function of Tyr91 was investigated in detail by comparing the crystal structures of mutants and their complexes with substrate analogs. In this study, tyrosine, a conserved amino acid near the active-site loop in the C-terminal domain of Sulfurisphaera tokodaii MGMT (StoMGMT), was mutated to phenylalanine to produce a Y91F mutant, and the cysteine which is responsible for receiving the methyl group in the active site was mutated to a serine to produce a C120S mutant. A Y91F/C120S double-mutant StoMGMT was also created. The function of tyrosine is discussed based on the crystal structure of Y91F mutant StoMGMT. The crystal structures of StoMGMT were determined at resolutions of 1.13-2.60 Å. They showed no structural changes except in the mutated part. No electron density for deoxyguanosine or methyl groups was observed in the structure of Y91F mutant crystals immersed in O6-methyl-2\'-deoxyguanosine, nor was the group oxidized in wild-type StoMGMT. Therefore, the hydroxy group of Tyr91 may prevent the oxidant from entering the active site. This suggests that tyrosine, which is highly conserved at the N-terminus of the helix-turn-helix motif across species, protects the active site of MGMTs, which are deactivated after repairing only one alkyl adduct. Overall, the results may provide a basis for understanding the molecular mechanisms by which high levels of conserved amino acids play a role in ensuring the integrity of suicide enzymes, in addition to promoting their activity.

Within the extremely thermoacidophilic Sulfolobaceae, the capacity to oxidize iron varies considerably. While some species are prolific iron oxidizers (e.g. Metallosphaera sedula), other species do not oxidize iron at all (e.g. Sulfolobus acidocaldarius). Iron oxidation capacity maps to a genomic locus, referred to previously as the \'Fox Cluster\', that encodes putative proteins that are mostly unique to the Sulfolobaceae. The role of putative proteins in the Fox Cluster has not been confirmed, but proteomic analysis here of iron-oxidizing membranes from M. sedula indicates that FoxA2 and FoxB (both cytochrome c oxidase-like subunits) and FoxC (CbsA/cytochrome b domain-containing) are essential. Furthermore, comparative genomics (locus organization and gene disruptions) and transcriptomics (polarity effects and differential expression) connect these genomic determinants with disparate iron biooxidation and respiration measurements among Sulfolobaceae species. While numerous homologous proteins can be identified for FoxA in genome databases (COX-like domains are prevalent across all domains of life), few homologues exist for FoxC or for most other Fox Cluster proteins. Phylogenetic reconstructions suggest this locus may have existed in early Sulfolobaceae, while the only other close homologues to the locus appear in the recently discovered candidate phylum Marsarchaota.

Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus, order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae, which is available at ictv.global/report/portogloboviridae.

Engineered heme enzymes such as myoglobin and cytochrome P450s metalloproteins are gaining widespread importance due to their efficiency in catalyzing non-natural reactions. In a recent strategy, the naturally occurring Fe metal in the heme unit was replaced with non-native metals such as Ir, Rh, Co, Cu, etc., and axial ligands to generate artificial metalloenzymes. Determining the best metal-ligand for a chemical transformation is not a trivial task. Here we demonstrate how computational approaches can be used in deciding the best metal-ligand combination which would be highly beneficial in designing new enzymes as well as small molecule catalysts. We have used Density Functional Theory (DFT) to shed light on the enhanced reactivity of an Ir system with varying axial ligands. We look at the insertion of a carbene group generated from diazo precursors via N2 extrusion into a C-H bond. For both Ir(Me) and Fe systems, the first step, i.e., N2 extrusion is the rate determining step. Strikingly, neither the better ligand overlap with 5d orbitals on Ir nor the electrophilicity on the carbene centre play a significant role. A comparison of Fe and Ir systems reveals that a lower distortion in the Ir(Me)-porphyrin on moving from the reactant to the transition state renders it catalytically more active. We notice that for both metal porphyrins, the free energy barriers are affected by axial ligand substitution. Further, for Fe porphyrin, the axial ligand also changes the preferred spin state. We show that for the carbene insertion into the C-H bond, Fe porphyrin systems undergo a stepwise HAT (hydrogen atom transfer) instead of a concerted hydride transfer process. Importantly, we find that the substitution of the axial Me ligand on Ir to imidazole or chloride, or without an axial substitution changes the rate determining step of the reaction. Therefore, an optimum ligand that can balance the barriers for both steps of the catalytic cycle is essential. We subsequently used the QM cluster approach to delineate the protein environment\'s role and mutations in improving the catalytic activity of the Ir(Me) system.