Archaeal Enzyme Research Articles

Common evolutionary origins of the bacterial glycyl tRNA synthetase and alanyl tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) establish the genetic code. Each aaRS covalently links a given canonical amino acid to a cognate set of tRNA isoacceptors. Glycyl tRNA aminoacylation is unusual in that it is catalyzed by different aaRSs in different lineages of the Tree of Life. We have investigated the phylogenetic distribution and evolutionary history of bacterial glycyl tRNA synthetase (bacGlyRS). This enzyme is found in early diverging bacterial phyla such as Firmicutes, Acidobacteria, and Proteobacteria, but not in archaea or eukarya. We observe relationships between each of six domains of bacGlyRS and six domains of four different RNA-modifying proteins. Component domains of bacGlyRS show common ancestry with (i) the catalytic domain of class II tRNA synthetases; (ii) the HD domain of the bacterial RNase Y; (iii) the body and tail domains of the archaeal CCA-adding enzyme; (iv) the anti-codon binding domain of the arginyl tRNA synthetase; and (v) a previously unrecognized domain that we call ATL (Ancient tRNA latch). The ATL domain has been found thus far only in bacGlyRS and in the universal alanyl tRNA synthetase (uniAlaRS). Further, the catalytic domain of bacGlyRS is more closely related to the catalytic domain of uniAlaRS than to any other aminoacyl tRNA synthetase. The combined results suggest that the ATL and catalytic domains of these two enzymes are ancestral to bacGlyRS and uniAlaRS, which emerged from common protein ancestors by bricolage, stepwise accumulation of protein domains, before the last universal common ancestor of life.

Occurrence, characteristics, and microbial community of microplastics in anaerobic sludge of wastewater treatment plants

Wastewater treatment plants (WWTPs) usually contain microplastics (MPs) due to daily influents of domestic and municipal wastewater. Thus, the WWTPs act as a point source of MPs distribution in the environment due to their incapability to remove MPs completely. In this study, MPs occurrence and distribution in anaerobic sludge from WWTPs in different regions (Kaifeng “KHP”, Jinan “JSP”, and Lanzhou “LGP”) were studied. Followed by MPs identification by microscopy and Fourier transform infrared (FTIR) spectrum. The microbial communities associated with anaerobic sludge and MPs were also explored. The results showed that MPs concentrations were 16.5, 38.5, and 17.2 particles/g of total solids (TS) and transparent MPs accounted for 49.1%, 58.5%, and 48.3% in KHP, JSP, and LGP samples, respectively. Fibers represented the most common shape of MPs in KHP (49.1%), JSP (56.0%), and LGP (69.0%). The FTIR spectroscopy indicated the predominance of polyethylene polymer in 1–5 mm MPs. The Proteobacteria, Chloroflexi, Actinobacteria, Bacteroidetes, and Planctomycetes were the abundant phyla in all anaerobic sludge. The bacterial genera in KHP and LGP were similar, in which Caldilinea (>23%), Terrimonas (>10%), and Ferruginibacter (>7%) formed the core bacterial genera. While Rhodococcus (15.3%) and Rhodoplanes (10.9%) were dominating in JSP. The archaeal genera Methanosaeta (>69%) and Methanobrevibacter (>10%) were abundant in KHP and LGP sludge. While Methanomethylovorans accounted for 90% of JSP. Acetyltransferase and hydratase were the major bacterial enzymes, while reductase was the key archaeal enzyme in all anaerobic sludge. This study provided the baseline for MPs distribution, characterization, and MPs associated microbes in WWTPs.

Structural basis for Tpt1-catalyzed 2'-PO4 transfer from RNA and NADP(H) to NAD.

Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2'-phosphate generated by tRNA ligase. Tpt1 also removes the 2'-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1″-PO4, ADP-ribose-2″-PO4, and 2'-OH RNA, and as substrate complexes with 2',5'-ADP and NAD+, that illuminate 2'-PO4 junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2'-PO4-containing metabolites NADP+ and NADPH as substrates for 2'-PO4 transfer to NAD+. A role in 2'-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2'-phosphorylation.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.

Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 μmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 μmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 μmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 μmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 μmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel.

To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces. The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy and Scanning Electron Microscopy (SEM) were used to evaluate cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis; and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS). Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.

Structural and biochemical elucidation of class I hybrid cluster protein natively extracted from a marine methanogenic archaeon.

Whilst widespread in the microbial world, the hybrid cluster protein (HCP) has been paradoxically a long-time riddle for microbiologists. During three decades, numerous studies on a few model organisms unravelled its structure and dissected its metal-containing catalyst, but the physiological function of the enzyme remained elusive. Recent studies on bacteria point towards a nitric oxide reductase activity involved in resistance during nitrate and nitrite reduction as well as host infection. In this study, we isolated and characterised a naturally highly produced HCP class I from a marine methanogenic archaeon grown on ammonia. The crystal structures of the enzyme in a reduced and partially oxidised state, obtained at a resolution of 1.45 and 1.36-Å, respectively, offered a precise picture of the archaeal enzyme intimacy. There are striking similarities with the well-studied enzymes from Desulfovibrio species regarding sequence, kinetic parameters, structure, catalyst conformations, and internal channelling systems. The close phylogenetic relationship between the enzymes from Methanococcales and many Bacteria corroborates this similarity. Indeed, Methanococcales HCPs are closer to these bacterial homologues than to any other archaeal enzymes. The relatively high constitutive production of HCP in M. thermolithotrophicus, in the absence of a notable nitric oxide source, questions the physiological function of the enzyme in these ancient anaerobes.

Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties

Although ADP-dependent sugar kinases were first described in archaea, at present, the presence of an ADP-dependent glucokinase (ADP-GK) in mammals is well documented. This enzyme is mainly expressed in hematopoietic lineages and tumor tissues, although its role has remained elusive. Here, we report a detailed kinetic characterization of the human ADP-dependent glucokinase (hADP-GK), addressing the influence of a putative signal peptide for endoplasmic reticulum (ER) destination by characterizing a truncated form. The truncated form revealed no significant impact on the kinetic parameters, showing only a slight increase in the Vmax value, higher metal promiscuity, and the same nucleotide specificity as the full-length enzyme. hADP-GK presents an ordered sequential kinetic mechanism in which MgADP is the first substrate to bind and AMP is the last product released, being the same mechanism described for archaeal ADP-dependent sugar kinases, in agreement with the protein topology. Substrate inhibition by glucose was observed due to sugar binding to nonproductive species.Although Mg2+ is an essential component for kinase activity, it also behaves as a partial mixed-type inhibitor for hADP-GK, mainly by decreasing the MgADP affinity. Regarding its distribution, phylogenetic analysis shows that ADP-GK's are present in a wide diversity of eukaryotic organisms although it is not ubiquitous. Eukaryotic ADP-GKs sequences cluster into two main groups, showing differences in the highly conserved sugar-binding motif reported for archaeal enzymes [NX(N)XD] where a cysteine residue is found instead of asparagine in a significant number of enzymes. Site directed mutagenesis of the cysteine residue by asparagine produces a 6-fold decrease in Vmax, suggesting a role for this residue in the catalytic process, probably by facilitating the proper orientation of the substrate to be phosphorylated.

Bacterial origins of thymidylate metabolism in Asgard archaea and Eukarya

Asgard archaea include the closest known archaeal relatives of eukaryotes. Here, we investigate the evolution and function of Asgard thymidylate synthases and other folate-dependent enzymes required for the biosynthesis of DNA, RNA, amino acids and vitamins, as well as syntrophic amino acid utilization. Phylogenies of Asgard folate-dependent enzymes are consistent with their horizontal transmission from various bacterial groups. We experimentally validate the functionality of thymidylate synthase ThyX of the cultured ‘Candidatus Prometheoarchaeum syntrophicum’. The enzyme efficiently uses bacterial-like folates and is inhibited by mycobacterial ThyX inhibitors, even though the majority of experimentally tested archaea are known to use carbon carriers distinct from bacterial folates. Our phylogenetic analyses suggest that the eukaryotic thymidylate synthase, required for de novo DNA synthesis, is not closely related to archaeal enzymes and might have been transferred from bacteria to protoeukaryotes during eukaryogenesis. Altogether, our study suggests that the capacity of eukaryotic cells to duplicate their genetic material is a sum of archaeal (replisome) and bacterial (thymidylate synthase) characteristics. We also propose that recent prevalent lateral gene transfer from bacteria has markedly shaped the metabolism of Asgard archaea.

Using several pseudo amino acid composition types and different machine learning algorithms to classify and predict archaeal phospholipases.

Phospholipases, as important lipolytic enzymes, have diverse industrial applications. Regarding the stability of extremophilic archaea's proteins in harsh conditions, analyses of unusual features of their proteins are significantly important for their utilization. This research was accomplished to in silico study of archaeal phospholipases' properties and to develop a pioneering method for distinguishing these enzymes from other archaeal enzymes via machine learning algorithms and Chou's pseudo-amino acid composition concept. The non-redundant sequences of archaeal phospholipases were collected. BioSeq-Analysis sever was used with Support Vector Machine (SVM), Random Forests (RF), Covariance Discrimination (CD), and Optimized Evidence-Theoretic K-nearest Neighbor (OET-KNN) as powerful machine learnings algorithms. Also, different Chou's pseudo-amino acid composition modes were performed and then, 5-fold cross-validation was applied to the sequences. Based on our results, the OET-KNN predictor, with 96% accuracy, yields the best performance in SC-PseAAC mode by 5-fold cross-validation. This predictor also achieved very high values of specificity (95%), sensitivity (96%), Matthews's correlation coefficient (0.92), and accuracy (96%). The present investigation yielded a robust anticipatory model for the archaeal phospholipase prediction utilizing the tenets PseAAC and OET-KNN machine learning algorithm.

Efficient biocatalytic C-H bond oxidation: an engineered heme-thiolate peroxygenase from a thermostable cytochrome P450.

A highly sought after reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds. We demonstrate the hydroxylation of fatty acids using an engineered thermostable archaeal cytochrome P450 enzyme. By replacing a seven amino acid section of the I-helix, the nicotinamide cofactor-dependent monooxygenase was converted into a hydrogen peroxide using peroxygenase, enabling the efficient biocatalytic oxidation of C-H bonds at room temperature to 90 °C.

Biochar addition augmented the microbial community and aided the digestion of high-loading slaughterhouse waste: Active enzymes of bacteria and archaea

The biogas production (BP), volatile fatty acids (VFAs), microbial communities, and microbes’ active enzymes were studied upon the addition of biochar (0–1.5%) at 6% and 8% slaughterhouse waste (SHW) loadings. The 0.5% biochar enhanced BP by 1.5- and 1.6-folds in 6% and 8% SHW-loaded reactors, respectively. Increasing the biochar up to 1.5% caused a reduction in BP at 6% SHW. However, the BP from 8% of SHW was enhanced by 1.4-folds at 1.5% biochar. The VFAs production in all 0.5% biochar amended reactors was highly significant compared to control (p-value < 0.05). The biochar addition increased the bacterial and archaeal diversity at both 6% and 8% SHW loadings. The highest number of OTUs at 0.5% biochar were 567 and 525 in 6% and 8% SHW, respectively. Biochar prompted the Clostridium abundance and increased the lyases and transaminases involved in the degradation of lipids and protein, respectively. Biochar addition improved the Methanosaeta and Methanosphaera abundance in which the major enzymes were reductase and hydrogenase. The archaeal enzymes showed mixed acetoclastic and hydrogenotrophic methanogenesis.

Waste cooking oils (WCOs) to biogas nexus: Kinetics, active microbes, and functional enzymes

The efficiency of anaerobic digestion (AD) chiefly relies on substrate composition and microbial structure. The lipidic substrates such as waste cooking oils (WCOs) have high biogas potential. The biogas production from WCOs can be increased by active microbes with simultaneous alleviation of environmental contamination. Thus, this study explored the biogas potential of two different WCOs including duck cooking oil (DCO) and fat, oil, and grease (FOG) in AD. The effects on biogas kinetics, oils degradation, microbial community dynamics, and variations in bacterial and archaeal functional enzymes were investigated. The modified Gompertz and logistic growth models showed a curve-fit within a range of 0.98–0.99 R2 value for biogas production in DCO and FOG-loaded reactors. The biogas production was > 5-folds higher in WCOs compared to control. The addition of DCO and FOG showed 76.87 % and 69.76 % of lipid degradation, respectively. The major bacterial enzymes involved in β-oxidation (based on Kyoto Encyclopaedia of Genes and Genomes, KEGG map 00071) of DCO and FOG were acetyl-CoA C-acetyltransferase and enoyl-CoA hydratase. These enzymes correspond to the dominance of Clostridium (>90 %) and might be involved in the formation of biomethane precursors. While the abundance of Methanosphaera (>60 %) and Methanobrevibacter (>30 %) increased the archaeal CoB-CoM reductases and methyltransferase involved in biomethanation. This study demonstrated that the WCOs could be used as potential substrates for biogas production.

A Nonconventional Archaeal Fluorinase Identified by In Silico Mining for Enhanced Fluorine Biocatalysis.

Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential catalysts for the biological synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorinases retrieved from systematic in silico mining and identified a nonconventional archaeal enzyme from Methanosaeta sp. that mediates the fastest SN2 fluorination rate reported to date. Furthermore, we demonstrate enhanced production of fluoronucleotides in vivo in a bacterial host engineered with this archaeal fluorinase, paving the way toward synthetic metabolism for efficient biohalogenation.

Fungi and Archaea Control Soil N2O Production Potential in Chinese Grasslands Rather Than Bacteria.

Nitrous oxide (N2O) is a powerful greenhouse gas and the predominant stratospheric ozone-depleting substance. Soil is a major source of N2O but remains largely uncertain due to the complicated processes of nitrification and denitrification performed by various groups of microbes such as bacteria, fungi, and archaea. We used incubation experiments to measure the total fungal, archaeal, and bacterial N2O production potential and the microbial functional genes in soils along 3,000 km Chinese grassland transect, including meadow steppe, typical steppe, desert steppe, alpine meadow, and alpine steppe. The results indicated that fungi, archaea, and bacteria contributed 25, 34, and 19% to nitrification and 46, 29, and 15% to denitrification, respectively. The AOA and AOB genes were notably correlated with the total nitrification enzyme activity (TNEA), whereas both narG and nirK genes were significantly correlated with total denitrification enzyme activity (TDEA) at p < 0.01. The correlations between AOA and ANEA (archaeal nitrification enzyme activity), AOB and BNEA (bacterial nitrification enzyme activity), and narG, nirK, and BDEA (bacterial denitrification enzyme activity) showed higher coefficients than those between the functional genes and TNEA/TDEA. The structural equation modeling (SEM) results showed that fungi are dominant in N2O production processes, followed by archaea in the northern Chinese grasslands. Our findings indicate that the microbial functional genes are powerful predictors of the N2O production potential, after distinguishing bacterial, fungal, and archaeal processes. The key variables of N2O production and the nitrogen (N) cycle depend on the dominant microbial functional groups in the N-cycle in soils.

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression.

Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by mcrABG) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F430. MCR houses a series of unusual post-translational modifications within its active site whose identities vary depending on the organism and whose functions remain unclear. Methanogenic MCRs are encoded in a highly conserved mcrBDCGA gene cluster, which encodes two accessory proteins, McrD and McrC, that are believed to be involved in the assembly and activation of MCR, respectively. The requirement of a unique and complex coenzyme, various unusual post-translational modifications, and many remaining questions surrounding assembly and activation of MCR largely limit in vitro experiments to native enzymes with recombinant methods only recently appearing. Production of MCRs in a heterologous host is an important step toward developing optimized biocatalytic systems for methane production as well as for bioconversion of methane and other alkanes into value-added compounds. This review will first summarize MCR catalysis and structure, followed by a discussion of advances and challenges related to the production of diverse MCRs in a heterologous host.

Nickel and the origin and early evolution of life.

Although nickel (Ni) is a minor element of the Earth's crust, it has played a major role in the evolution of life. This metal is a component of the active sites of several archaeal and bacterial anaerobic enzymes essential for bioenergy processes such as H2 and CO oxidation and CO2 fixation. Furthermore, Ni of meteoritic origin was probably involved in primordial organic phosphorylations. However, depending on its concentration, Ni can also be extremely toxic to most species. Through Earth's history, this paradoxical situation has provoked complex interactions between microorganisms, such as sulfate-reducing bacteria and the highly Ni-dependent methanogens. Ni-rich volcanic emissions have resulted in alterations of the biological carbon cycle caused by high archaeal production of greenhouse CH4 gas and the ensuing global temperature elevation. These emissions are also thought to have directly helped producing the most serious of the five major extinctions at the end of the Permian period.

Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions.

Agricultural methane produced by archaea in the forestomach of ruminants is a key contributor to rising levels of greenhouse gases leading to climate change. Functionalized biological polyhydroxybutyrate (PHB) nanoparticles offer a new concept for the reduction of enteric methane emissions by inhibiting rumen methanogens. Nanoparticles were functionalized in vivo with an archaeal virus lytic enzyme, PeiR, active against a range of rumen Methanobrevibacter species. The impact of functionalized nanoparticles against rumen methanogens was demonstrated in pure cultures, in rumen batch and continuous flow rumen models yielding methane reduction of up to 15% over 11 days in the most complex system. We further present evidence of biological nanoparticle fermentation in a rumen environment. Elevated levels of short-chain fatty acids essential to ruminant nutrition were recorded, giving rise to a promising new strategy combining methane mitigation with a possible increase in animal productivity.

High electrochemical stability of hyperthermophilic archaeal multicopper enzyme adsorbed on gold electrodes compared to fungal laccase

Multicopper enzymes (MCEs) have attracted significant recent attention for use as biocathodes in enzyme-catalyzed fuel cells, mostly to facilitate the four-electron reduction of oxygen to water. Au is highly biocompatible and does not exhibit a carbon-like tendency for toxicity. Especially, the development of an implant-type enzyme fuel cells with noble electrodes, such as those made of Au, can assist in resolving health issues. In the present research, a direct electron transfer (DET)-type reaction featuring a hyperthermophilic archaeon multicopper enzyme (McoP) and a Trametes sp. laccase (Lac) adsorbed on bare Au electrodes was investigated. Quartz crystal microbalance (QCM) analysis was used to examine the adsorption behavior of McoP and Lac on Au electrodes. The obtained results indicated that the adsorbed McoP on Au exhibited stability against a higher potential electric field (+0.8 V vs. Ag|AgCl) and showed stable bioelectrocatalysis for oxygen reduction (main production of H2O2 with two-electron reaction), compared with Lac. The electron transfer rate of the McoP at the Au electrode interface was evaluated to be 10 s−1. This finding could urgently contribute for developing biocathodes in enzyme-catalyzed fuel cell, especially implant-type enzymatic fuel cells.

Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6.

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Å resolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.