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Published version. Source at http://dx.doi.org/10.1016/j.csbj.2016.04.001 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 froma decade of structural and functional studies of the DNA repairmachinery of Deinococcus radiodurans and discuss howthese 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.

Our cellular macromolecules are continuously damaged by harmful reactive oxygen species (ROS) which are produced as natural by-products during intracellular processes (respiration) and by external sources (e.g. UV-irradiation and cigarette smoke). If these damages are not prevented and/or repaired, they will promote oxidative damages which ultimately can lead to development of cancer, neurological diseases and premature aging. Fortunately, the presence of redox scavenging systems and DNA repair machineries reduces the extent of induced damages; however, the underlying molecular mechanisms of these systems are largely unknown. Here we describe the cloning and expression tests of one peroxidase – Dye decolorizing peroxidase - from the extremely radiation and desiccation resistant bacterium Deinococcus radiodurans, in order to determine its potential role as part of the antioxidant defense system. The enzyme was overexpressed in E.coli, purified, crystallised and characterized by UV-vis and Resonance Raman spectroscopies. Our aim is to perform structure/function analysis of this protein and in a long term prespective, improve our understanding of oxidative stress related human diseases.

The genes encoding uracil-DNA N -glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida and the mesophilic counterpart Vibrio cholerae have been cloned and expressed in Escherichia coli . The purified proteins have been characterized in order to reveal possible cold adapted features of the V. salmonicida UNG (vsUNG) compared to the V. cholerae UNG (vcUNG). Characterization experiments demonstrated that both enzymes possessed the highest activities at pH 7.0–7.5 and at salt concentrations in the range of 25–50 mM NaCl. Temperature optima for activity were determined to approximately 30 °C for vsUNG and 50 °C for vcUNG. Temperature stability of the enzymes was compared at 4 °C and 37 °C, and vsUNG was found to be more temperature labile than vcUNG. Kinetic studies performed at three different temperatures, 15 °C, 22 °C and 37 °C, demonstrated higher catalytic efficiency for vsUNG compared to vcUNG due to lower K M -values. The increased substrate affinity of vsUNG is probably caused by an increased number of positively charged residues in the DNA-binding site of the enzyme compared to vcUNG. Thus, activity and stability measurements reveal typical cold adapted features of vsUNG.

The periplasmic/extracellular bacterial enzyme endonuclease I was chosen as a model system to identify features that might be responsible for temperature- and salt adaptation. A statistical study of amino acid sequence properties belonging to endonuclease I enzymes from three mesophilic habitats (non-marine, brackish water and marine), and three marine temperature groups (psychrophile, intermediate and mesophile) has been conducted. Ten new endonuclease I genes have been sequenced in order to increase the sample size. A bioinformatical method of property dependent statistical analysis of alignments has been applied. To our knowledge this is the first time these methods have been used in order to investigate environmental adaptation of enzymes. Adaptation to low temperature seems to involve increased surface isoelectric point and hydrophobicity in contrast to salt adaptation in which the isoelectric point and hydrophobicity at the surface decreases. Redistribution of charge and hydrophobicity might be the most important signature for cold adaptation and salt adaptation of this enzyme class. The results indicate that general trends of adaptation are possible to elucidate from the amino acid sequences. Also in this paper a new scale of stratified B-factors, derived from the Protein Data Bank, is presented.

Deinococcus radiodurans is extremely resistant to the effects of ionizing radiation. The source of the radiation resistance is not known, but an expansion of specific protein families related to stress response and damage control has been observed. DNA repair enzymes are among the expanded protein families in D. radiodurans, and genes encoding five different uracil-DNA glycosylases are identified in the genome. Here we report the three-dimensional structure of the mismatch-specific uracil-DNA glycosylase (MUG) from D. radiodurans (drMUG) to a resolution of 1.75 angstroms. Structural analyses suggest that drMUG possesses a novel catalytic residue, Asp-93. Activity measurements show that drMUG has a modified and broadened substrate specificity compared with Escherichia coli MUG. The importance of Asp-93 for activity was confirmed by structural analysis and abolished activity for the mutant drMUGD93A. Two other microorganisms, Bradyrhizobium japonicum and Rhodopseudomonas palustris, possess genes that encode MUGs with the highest sequence identity to drMUG among all of the bacterial MUGs examined. A phylogenetic analysis indicates that these three MUGs form a new MUG/thymidine-DNA glycosylase subfamily, here called the MUG2 family. We suggest that the novel catalytic residue (Asp-93) has evolved to provide drMUG with broad substrate specificity to increase the DNA repair repertoire of D. radiodurans.

Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway and removes uracil from the DNA strand. Atlantic cod UDG (cUDG), which is a cold-adapted enzyme, has been found to be up to 10 times more catalytically active in the temperature range 15-37 °C as compared with the warm-active human counterpart. The increased catalytic activity of cold-adapted enzymes as compared with their mesophilic homologues are partly believed to be caused by an increase in the structural flexibility. However, no direct experimental evidence supports the proposal of increased flexibility of cold-adapted enzymes. We have used molecular dynamics simulations to gain insight into the structural flexibility of UDG. The results from these simulations show that an important loop involved in DNA recognition (the Leu272 loop) is the most flexible part of the cUDG structure and that the human counterpart has much lower flexibility in the Leu272 loop. The flexibility in this loop correlates well with the experimental kcat/Km values. Thus, the data presented here add strong support to the idea that flexibility plays a central role in adaptation to cold environments.

The present work investigates the possibility of constructing a multivariate calibration model for predicting the composition of ground beef with respect to different meat quality types, based on intensity profiles from isoelectric focusing of water-soluble proteins. Beef mixtures containing various amounts of mechanically recovered meat, head meat and production meat from beef, were analysed by isoelectric focusing in immobilised pH-gradients. The gels were photographed and the images transferred to a digital format. By simple image processing procedures, background colour was virtually eliminated and signal strength was improved to a considerable degree. Multivariate analysis of protein profiles from the gels gave models explaining 75 to 90% of variance in sample composition. Manually deboned meat was explained to the highest degree, and with a precision of 7%. Two different qualities of mechanically recovered meat could be detected even when treated as one category. The present approach needs further refinement, but seems applicable for detecting intentional substitution of high quality meat products with low-price raw materials. One advantage of the approach is that evaluation of samples is not dependent on specific knowledge on the individual components to be analysed, so that such analytical methods are relatively easy to implement in any standard laboratory.

Saccharomyces cerevisiae was used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFα promoter to the inducible CUP1 promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS–PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin gene SSO2 , but the total level of the hormone was not improved due to impaired growth.