Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system.

Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, which was prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridge. Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52kDa proenzyme was processed at K137 and K144 into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H132/C185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system.

Deep-sea hydrothermal vent systems with prevailing extreme thermal conditions for life offer unique habitats to source heat tolearant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain , prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid- Ocean Ridges. By sequence comparisons against the MEROPS-MPRO database, globupain showed highest sequence identity to C11-like proteases present in human gut and intestinal bacteria,. Successful recombinant expression in Escherichia coli of the active zymogen and 13 mutant substitution variants allowed assesment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca²âº. When activated, the 52 kDa proenzyme was processed at Lys 137 and Lys 144 into a 12 kDa light- and 32 kDa heavy chain heterodimer. A structurally conserved His 132 /Cys 185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans . Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR- aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (T m activated enzyme = 94.51 ± 0.09°C) with optimal activity at 75 °C and pH 7.1. By characterizing globupain, our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases have been expanded. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors.

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/ß-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3'-5' exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3'-5' exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme's activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme's moderate thermal stability.

Going to extremes - a metagenomic journey into the dark matter of life.

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.

Ts2631 Endolysin from the Extremophilic Thermus scotoductus Bacteriophage vB_Tsc2631 as an Antimicrobial Agent against Gram-Negative Multidrug-Resistant Bacteria.

Bacteria that thrive in extreme conditions and the bacteriophages that infect them are sources of valuable enzymes resistant to denaturation at high temperatures. Many of these heat-stable proteins are useful for biotechnological applications; nevertheless, none have been utilized as antibacterial agents. Here, we demonstrate the bactericidal potential of Ts2631 endolysin from the extremophilic bacteriophage vB_Tsc2631, which infects Thermus scotoductus, against the alarming multidrug-resistant clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa and pathogens from the Enterobacteriaceae family. A 2-3.7 log reduction in the bacterial load was observed in antibacterial tests against A. baumannii and P. aeruginosa after 1.5 h. The Ts2631 activity was further enhanced by ethylenediaminetetraacetic acid (EDTA), a metal ion chelator (4.2 log reduction in carbapenem-resistant A. baumannii) and, to a lesser extent, by malic acid and citric acid (2.9 and 3.3 log reductions, respectively). The EDTA/Ts2631 combination reduced all pathogens of the Enterobacteriaceae family, particularly multidrug-resistant Citrobacter braakii, to levels below the detection limit (>6 log); these results indicate that Ts2631 endolysin could be useful to combat Gram-negative pathogens. The investigation of A. baumannii cells treated with Ts2631 endolysin variants under transmission electron and fluorescence microscopy demonstrates that the intrinsic antibacterial activity of Ts2631 endolysin is dependent on the presence of its N-terminal tail.

Structure and function of the Ts2631 endolysin of Thermus scotoductus phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding.

To escape from hosts after completing their life cycle, bacteriophages often use endolysins, which degrade bacterial peptidoglycan. While mesophilic phages have been extensively studied, their thermophilic counterparts are not well characterized. Here, we present a detailed analysis of the structure and function of Ts2631 endolysin from thermophilic phage vB_Tsc2631, which is a zinc-dependent amidase. The active site of Ts2631 consists of His30, Tyr58, His131 and Cys139, which are involved in Zn2+ coordination and catalysis. We found that the active site residues are necessary for lysis yet not crucial for peptidoglycan binding. To elucidate residues involved in the enzyme interaction with peptidoglycan, we tested single-residue substitution variants and identified Tyr60 and Lys70 as essential residues. Moreover, substitution of Cys80, abrogating disulfide bridge formation, inactivates Ts2631, as do substitutions of His31, Thr32 and Asn85 residues. The endolysin contains a positively charged N-terminal extension of 20 residues that can protrude from the remainder of the enzyme and is crucial for peptidoglycan binding. We show that the deletion of 20 residues from the N-terminus abolished the bacteriolytic activity of the enzyme. Because Ts2631 exhibits intrinsic antibacterial activity and unusual thermal stability, it is perfectly suited as a scaffold for the development of antimicrobial agents.