The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). . Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

Uracil-DNA glycosylase (UDG; EC 3.2.2.3) is a DNA-repair protein that catalyses the hydrolysis of promutagenic uracil residues from single- or double-stranded DNA, generating free uracil and abasic DNA. The crystal structure of the catalytic domain of cod uracil-DNA glycosylase (cUDG) has been determined to 1.9 A resolution, with final R factors of 18.61 and 20.57% for the working and test sets of reflections, respectively. This is the first crystal structure of a uracil-DNA glycosylase from a cold-adapted species and a detailed comparison with the human enzyme is performed in order to rationalize the cold-adapted behaviour of the cod enzyme at the structural level. The catalytic domain of cUDG comprises 223 residues, with a sequence identity to the human UDG of 75%. The tertiary structures of the two enzymes are also similar, with an overall displacement in main-chain atomic positions of 0.63 A. The amino-acid substitutions and the differences in intramolecular hydrogen bonds, hydrophobic interactions, ion-pair interactions and electrostatic potentials are compared and discussed in order to gain insight into the factors that cause the increased activity and reduced thermostability of the cod enzyme. In particular, the reduced number of strong ion-pair interactions in the C-terminal half of cUDG is believed to greatly affect the flexibility and/or stability. Increased positive electrostatic surface potential on the DNA-facing side of cUDG seems to be responsible for increasing the affinity for the negatively charged DNA compared with that of hUDG.

The marine and estuarine environment harbors a vast diversity of bacteria. Some of the most extensively studied marine or estuarine bacteria belong to the genus Vibrio, with Vibrio cholerae being the most notorious species as it is the cause of cholera in humans. V. cholerae is found in tropical and temperate areas, and can be classified as a mesophilic bacterium with growth optimum around 37°C. It prefers estuarine waters, is halotolerant, and does not require NaCl for growth [1,2]. The bacterium with one of the lowest growth optimum temperatures found in the genus Vibrio is the fish pathogen Vibrio salmonicida. It has an optimal growth temperature of % 15°C and requires NaCl for growth [3]. It can therefore be classified as a psychrophilic and mildly halophilic bacterium.A living cell can be considered as a chemical factory which produces many substances. The speed of production is limited by reaction rates. The reaction rates are in turn limited by, among other things, environmental factors such as pH, salinity, pressure and temperature. Temperature is a very important factor for growth and proliferation of the cells. At high temperatures, at which thermophiles thrive, chemical reaction rates are very high, and the main challenge for cells is to adapt their enzymes, membranes and molecules to cope with the heat. At low temperatures, the chemical reaction rates are lower, and hence, in order to be competitive and grow fast at low temperatures, evolutionary pressure favors enzymes that are more efficient than their high-temperature counterparts. This higher efficiency at low temperatures is believed to be caused Endonuclease I is a periplasmic or extracellular enzyme present in many different Proteobacteria. The endA gene encoding endonuclease I from the psychrophilic and mildly halophilic bacterium Vibrio salmonicida and from the mesophilic brackish water bacterium Vibrio cholerae have been cloned, over-expressed in Escherichia coli, and purified. A comparison of the enzymatic properties shows large differences in NaCl requirements, optimum pH, temperature stability and catalytic efficiency of the two proteins. The V. salmonicida EndA shows typical cold-adapted features such as lower unfolding temperature, lower temperature optimum for activity, and higher specific activity than V. cholerae EndA. The thermodynamic activation parameters confirm the psychrophilic nature of V. salmonicida EndA with a much lower activation enthalpy. The optimal conditions for enzymatic activity coincide well with the corresponding optimal requirements for growth of the organisms, and the enzymes function predominantly as DNases at physiological concentrations of NaCl. The periplasmic or extracellular localization of the enzymes, which renders them constantly exposed to the outer environment of the cell, may explain this fine-tuning of biochemical properties.Abbreviations DSC, differential scanning calorimetry; VcEndA, recombinant Vibrio cholerae endonuclease I; VsEndA, recombinant Vibrio salmonicida endonuclease I.

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N 6 -ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several sitedirected mutants, forms a molecular rationalization for the newly discovered base excision activity.

No abstract

Uracil-DNA glycosylases are DNA-repair enzymes that catalyse the removal of promutagenic uracil from single- and double-stranded DNA, thereby initiating the base-excision repair (BER) pathway. Uracil in DNA can occur by mis-incorporation of dUMP in place of dTMP during DNA synthesis or by deamination of cytosine, resulting in U-A or U-G mispairs. The radiation-resistant bacterium Deinococcus radiodurans has an elevated number of uracil-DNA glycosylases compared with most other organisms. The crystal structure of dr0689 (uracil-DNA N-glycosylase), which has been shown to be the major contributor to the removal of mis-incorporated uracil bases in crude cell extracts of D. radiodurans, is reported.

The Deinococcus radiodurans bacterium is extremely resistant to ionising radiation and desiccation and can withstand a 200-fold higher radiation dose than most other bacteria with no loss of viability. The mechanisms behind this extreme resistance are not fully understood, but it is clear that several factors contribute to this phenotype. Efficient scavenging of reactive oxygen species and repair of damaged DNA are two of these. In this review, we summarise the results from a decade of structural and functional studies of the DNA repair machinery of Deinococcus radiodurans and discuss how these studies have contributed to an improved understanding of the molecular mechanisms underlying DNA repair and to the outstanding resistance of Deinococcus radiodurans to DNA damaging agents.