Draft Genome Sequence of the Symbiotically Competent Cyanobacterium Nostoc sp. Strain KVJ20.

Nostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla This cyanobacterium has been shown to have broad symbiotic competence, and bacterial extracts have inhibitory effects on cancer cell lines and microbes. An array of genes for the production of secondary metabolites is present.

Features and structure of a cold active N-acetylneuraminate lyase.

N-acetylneuraminate lyases (NALs) are enzymes that catalyze the reversible cleavage and synthesis of sialic acids. They are therefore commonly used for the production of these high-value sugars. This study presents the recombinant production, together with biochemical and structural data, of the NAL from the psychrophilic bacterium Aliivibrio salmonicida LFI1238 (AsNAL). Our characterization shows that AsNAL possesses high activity and stability at alkaline pH. We confirm that these properties allow for the use in a one-pot reaction at alkaline pH for the synthesis of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) from the inexpensive precursor N-acetylglucosamine. We also show that the enzyme has a cold active nature with an optimum temperature for Neu5Ac synthesis at 20°C. The equilibrium constant for the reaction was calculated at different temperatures, and the formation of Neu5Ac acid is favored at low temperatures, making the cold active enzyme a well-suited candidate for use in such exothermic reactions. The specific activity is high compared to the homologue from Escherichia coli at three tested temperatures, and the enzyme shows a higher catalytic efficiency and turnover number for cleavage at 37°C. Mutational studies reveal that amino acid residue Asn 168 is important for the high kcat. The crystal structure of AsNAL was solved to 1.65 Å resolution and reveals a compact, tetrameric protein similar to other NAL structures. The data presented provides a framework to guide further optimization of its application in sialic acid production and opens the possibility for further design of the enzyme.

Structure and function of a CE4 deacetylase isolated from a marine environment.

Chitin, a polymer of ß(1-4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.

Isolation and complete genome sequence of the thermophilic Geobacillus sp. 12AMOR1 from an Arctic deep-sea hydrothermal vent site.

Members of the genus Geobacillus have been isolated from a wide variety of habitats worldwide and are the subject for targeted enzyme utilization in various industrial applications. Here we report the isolation and complete genome sequence of the thermophilic starch-degrading Geobacillus sp. 12AMOR1. The strain 12AMOR1 was isolated from deep-sea hot sediment at the Jan Mayen hydrothermal Vent Site. Geobacillus sp. 12AMOR1 consists of a 3,410,035 bp circular chromosome and a 32,689 bp plasmid with a G + C content of 52 % and 47 %, respectively. The genome comprises 3323 protein-coding genes, 88 tRNA species and 10 rRNA operons. The isolate grows on a suite of sugars, complex polysaccharides and proteinous carbon sources. Accordingly, a versatility of genes encoding carbohydrate-active enzymes (CAZy) and peptidases were identified in the genome. Expression, purification and characterization of an enzyme of the glycoside hydrolase family 13 revealed a starch-degrading capacity and high thermal stability with a melting temperature of 76.4 °C. Altogether, the data obtained point to a new isolate from a marine hydrothermal vent with a large bioprospecting potential.

Structural and functional characterization of two unusual endonuclease III enzymes from Deinococcus radiodurans.

While most bacteria possess a single gene encoding the bifunctional DNA glycosylase Endonuclease III (EndoIII) in their genomes, Deinococcus radiodurans possesses three: DR2438 (DrEndoIII1), DR0289 (DrEndoIII2) and DR0982 (DrEndoIII3). Here we have determined the crystal structures of DrEndoIII1 and an N-terminally truncated form of DrEndoIII3 (DrEndoIII3Δ76). We have also generated a homology model of DrEndoIII2 and measured activity of the three enzymes. All three structures consist of two all α-helical domains, one of which exhibits a [4Fe-4S] cluster and the other a HhH-motif, separated by a DNA binding cleft, similar to previously determined structures of endonuclease III from Escherichia coli and Geobacillus stearothermophilus. However, both DrEndoIII1 and DrEndoIII3 possess an extended HhH motif with extra helical features and an altered electrostatic surface potential. In addition, the DNA binding cleft of DrEndoIII3 seems to be less accessible for DNA interactions, while in DrEndoIII1 it seems to be more open. Analysis of the enzyme activities shows that DrEndoIII2 is most similar to the previously studied enzymes, while DrEndoIII1 seems to be more distant with a weaker activity towards substrate DNA containing either thymine glycol or an abasic site. DrEndoIII3 is the most distantly related enzyme and displays no detectable activity towards these substrates even though the suggested catalytic residues are conserved. Based on a comparative structural analysis, we suggest that the altered surface potential, shape of the substrate-binding pockets and specific amino acid substitutions close to the active site and in the DNA interacting loops may underlie the unexpected differences in activity.

Characterization of the N-acetylneuraminic acid synthase (NeuB) from the psychrophilic fish pathogen Moritella viscosa.

Moritella viscosa is a Gram-negative psychrophilic bacterium that causes winter ulcer disease in Atlantic salmon and cod. Its genome reveals that it possesses the ability to synthesize sialic acids. Indeed, sialic acid can be isolated from the bacterium and when analyzed using HPLC-MS/MS, the presence of N-acetylneuraminic acid was confirmed. Thus, the N-acetylneuraminic acid synthase NeuB from M. viscosa (MvNeuB) was recombinantly produced and characterized. The optimum pH and temperature for MvNeuB activity are 7.5 and 30 °C, respectively. The KM for N-acetylmannosamine and phosphoenolpyruvate is 18±5 and 0.8±0.2 mM, respectively. The kcat value (∼225 min(-1)) for both N-acetylmannosamine and phosphoenolpyruvate is the highest turnover number found for an enzyme in this class until the date. A calorimetric study of MvNeuB shows that the enzyme has a two-step transition peak probably reflecting the two domains these proteins consist of. MvNeuB is less stable at higher temperature and has a high catalytic activity at lower temperature compared to mesophilic counterparts. Enzymes from psychrophilic organisms are generally cold adapted meaning they can maintain adequate function near the freezing point of water. Cold adapted enzymes are catalytically more efficient at lower temperature and are more thermo-labile compared to their mesophilic counterparts. MvNeuB is a typical cold adapted enzyme and could be further explored for production of sialic acids and derivates at low temperatures.

High quality draft genome sequence of Streptomyces sp. strain AW19M42 isolated from a sea squirt in Northern Norway.

Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.

Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi).

Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme-substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG-Ugi was determined to a resolution of 1.9 Šwith eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG-Ugi complex with previously determined structures of UNG-Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (Kb) which is one order of magnitude larger than that for hUNG-Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein-Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.

Characterization of the sialic acid synthase from Aliivibrio salmonicida suggests a novel pathway for bacterial synthesis of 7-O-acetylated sialic acids.

Resolving the enzymatic pathways leading to sialic acids (Sias) in bacteria are vitally important for understanding their roles in pathogenesis and for subsequent development of tools to combat infections. A detailed characterization of the involved enzymes is also essential due to the highly applicable properties of Sias, i.e., as used in a wide range of medical applications and human nutrition. Bacterial strains that produce Sias display them mainly on their cell surface to mimic animal cells thereby evading the host's immune system. Despite several studies, little is known about the virulence mechanisms of the fish pathogen Aliivibrio salmonicida. The genome of A. salmonicida LFI1238 contains a gene cluster homologous to the Escherichia coli neuraminic acid (Neu) gene cluster involved in biosynthesis of Sias found in the E. coli capsule. This cluster is probably responsible for the biosynthesis of Neu found in A. salmonicida. In this work, we have produced and characterized the sialic acid (Sia) synthase NeuB1, the key enzyme in the pathway. The Sia synthase is an enzyme producing N-acetylneuraminic acid by the condensation of N-acetylmannosamine and phosphoenolpyruvate. Genome content, kinetic data obtained, together with structural considerations, have led us to the prediction that the substrate for NeuB1 from A. salmonicida, E. coli and Streptococcus agalactiae among others, is 4-O-acetyl-N-acetylmannosamine. This means that the product of its enzymatic reaction is 7-O-acetyl-N-acetylneuraminic acid. We propose a pathway for production of this Sia in A. salmonicida, and present evidence for the presence of diacetylated Neu in the bacterium.

Draft Genome Sequence of the Actinomycete Rhodococcus sp. Strain AW25M09, Isolated from the Hadsel Fjord, Northern Norway.

The cold-adapted Rhodococcus sp. strain AW25M09 was isolated from an Atlantic hagfish caught off the shore of northern Norway as part of an ongoing bioprospecting project that aims to identify novel bacteria with biotechnological potential. Here, we present the 5.8-Mb draft genome sequence, together with details regarding the origin of the strain and its sequence assembly.

Thermal unfolding studies of cold adapted uracil-DNA N-glycosylase (UNG) from Atlantic cod (Gadus morhua). A comparative study with human UNG.

Uracil-DNA N-glycosylase (UNG; EC 3.2.2.27) from Atlantic cod (cUNG) possesses cold adapted features like increased catalytic efficiency and reduced temperature optimum for activity compared to its warm-adapted homologue human UNG (hUNG). Here, we present the first thermal stability analysis of cUNG and hUNG by differential scanning calorimetry (DSC), and the results showed that cUNG is less stable than hUNG and unfolds at a melting temperature (T(m)) 9° lower than its warm-adapted homologue. In addition, an ion-pair (D183-K302) suggested to be crucial for global stability of hUNG was investigated by biochemical characterization and DSC of four mutants (cUNG G183D and cUNG G183D-R302K, hUNG D183G and hUNG D183G-K302R). The hUNG mutants with an expected disruption of the ion-pair showed a slight increase in stability with concomitant reduction in the enzyme activity, while the apparent introduction of the ion-pair in cUNG caused a reduction in the enzyme activity but no increase in stability. Because the mutants did not behave as expected, the phenomenon was further investigated by crystal structure determination. Indeed, the crystal structure of the hUNG D183G-K302R mutant revealed that compensating interactions for the loss of the ion-pair were generated close to and in regions distant from the mutation site. In conclusion, the reduced stability of cUNG supports the suggested requirement of a flexible structure for improved activity at low temperatures. Furthermore, the lack of a direct correlation between enzyme activity and global stability of the mutants supports the significance of distributing locally flexible and/or rigid regions for modulation of enzyme activity.

Thermodynamics and structure of a salmon cold active goose-type lysozyme.

Atlantic salmon goose-type lysozyme (SalG) was previously shown to display features of cold-adaptation as well as renaturation following heat treatment. In this study differential scanning calorimetry (DSC) was carried out to investigate unfolding and potential refolding, while X-ray crystallography was used to study structural factors contributing to the temperature-related characteristics. The recombinant SalG has a melting temperature (T(m)) of 36.8 degrees C under thermal denaturation conditions and regains activity after returning to permissive (low) temperature. Furthermore, refolding is dramatically reduced in solutions with high SalG concentrations, coupled with significant protein precipitation. The structural features of SalG closely resemble those of other g-type lysozymes. However, the N-terminal region of SalG is less anchored to the rest of the molecule due to the absence of disulphide bonds, thus, contributing significantly to the low T(m) of SalG. The absence of disulphide bonds and the distribution of salt bridges may at the same time ease refolding leading to renaturation.

Structure of uracil-DNA N-glycosylase (UNG) from Vibrio cholerae: mapping temperature adaptation through structural and mutational analysis.

The crystal structure of Vibrio cholerae uracil-DNA N-glycosylase (vcUNG) has been determined to 1.5 A resolution. Based on this structure, a homology model of Aliivibrio salmonicida uracil-DNA N-glycosylase (asUNG) was built. A previous study demonstrated that asUNG possesses typical cold-adapted features compared with vcUNG, such as a higher catalytic efficiency owing to increased substrate affinity. Specific amino-acid substitutions in asUNG were suggested to be responsible for the increased substrate affinity and the elevated catalytic efficiency by increasing the positive surface charge in the DNA-binding region. The temperature adaptation of these enzymes has been investigated using structural and mutational analyses, in which mutations of vcUNG demonstrated an increased substrate affinity that more resembled that of asUNG. Visualization of surface potentials revealed a more positive potential for asUNG compared with vcUNG; a modelled double mutant of vcUNG had a potential around the substrate-binding region that was more like that of asUNG, thus rationalizing the results obtained from the kinetic studies.

Structure of a highly stable mutant of human fibroblast growth factor 1.

Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).

Structural evidence for lack of inhibition of fish goose-type lysozymes by a bacterial inhibitor of lysozyme.

It is known that bacteria contain inhibitors of lysozyme activity. The recently discovered Escherichia coli inhibitor of vertebrate lysozyme (Ivy) and its potential interactions with several goose-type (g-type) lysozymes from fish were studied using functional enzyme assays, comparative homology modelling, protein-protein docking, and molecular dynamics simulations. Enzyme assays carried out on salmon g-type lysozyme revealed a lack of inhibition by Ivy. Detailed analysis of the complexes formed between Ivy and both hen egg white lysozyme (HEWL) and goose egg white lysozyme (GEWL) suggests that electrostatic interactions make a dominant contribution to inhibition. Comparison of three dimensional models of aquatic g-type lysozymes revealed important insertions in the beta domain, and specific sequence substitutions yielding altered electrostatic surface properties and surface curvature at the protein-protein interface. Thus, based on structural homology models, we propose that Ivy is not effective against any of the known fish g-type lysozymes. Docking studies suggest a weaker binding mode between Ivy and GEWL compared to that with HEWL, and our models explain the mechanistic necessity for conservation of a set of residues in g-type lysozymes as a prerequisite for inhibition by Ivy.

Structural adaptation of endonuclease I from the cold-adapted and halophilic bacterium Vibrio salmonicida.

The crystal structure of the periplasmic/extracellular endonuclease I from Vibrio salmonicida has been solved to 1.5 A resolution and, in comparison to the corresponding endonucleases from V. cholerae and V. vulnificus, serves as a model system for the investigation of the structural determinants involved in the temperature and NaCl adaptation of this enzyme class. The overall fold of the three enzymes is essentially similar, but the V. salmonicida endonuclease displays a significantly more positive surface potential than the other two enzymes owing to the presence of ten more Lys residues. However, if the optimum salt concentrations for the V. salmonicida and V. cholerae enzymes are taken into consideration in the electrostatic surface-potential calculation, the potentials of the two enzymes become surprisingly similar. The higher number of basic residues in the V. salmonicida protein is therefore likely to be a result, at least in part, of adaptation to the more saline habitat of V. salmonicida (seawater) than V. cholerae (brackish water). The hydrophobic core of all three enzymes is almost identical, but the V. salmonicida endonuclease has a slightly lower number of internal hydrogen bonds. This, together with repulsive forces between the basic residues on the protein surface of V. salmonicida endonuclease I and differences in the distribution of salt bridges, probably results in higher flexibility of regions of the V. salmonicida protein. This is likely to influence both the catalytic activity and the stability of the protein.

Effects of salt on the kinetics and thermodynamic stability of endonuclease I from Vibrio salmonicida and Vibrio cholerae.

Adaptation to extreme environments affects the stability and catalytic efficiency of enzymes, often endowing them with great industrial potential. We compared the environmental adaptation of the secreted endonuclease I from the cold-adapted marine fish pathogen Vibrio salmonicida (VsEndA) and the human pathogen Vibrio cholerae (VcEndA). Kinetic analysis showed that VsEndA displayed unique halotolerance. It retained a considerable amount of activity from low concentrations to at least 0.6 m NaCl, and was adapted to work at higher salt concentrations than VcEndA by maintaining a low K(m) value and increasing k(cat). In differential scanning calorimetry, salt stabilized both enzymes, but the effect on the calorimetric enthalpy and cooperativity of unfolding was larger for VsEndA, indicating salt dependence. Mutation of DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters. The VsEndA Q69N mutation also increased the T(m) value, whereas other mutations affected mainly DeltaH(cal). The determined crystal structure of VcEndA N69Q revealed the loss of one hydrogen bond present in native VcEndA, but also the formation of a new hydrogen bond involving residue 69 that could possibly explain the similar T(m) values for native and N69Q-mutated VcEndA. Structural analysis suggested that the stability, catalytic efficiency and salt tolerance of EndA were controlled by small changes in the hydrogen bonding networks and surface electrostatic potential. Our results indicate that endonuclease I adaptation is closely coupled to the conditions of the habitats of natural Vibrio, with VsEndA displaying a remarkable salt tolerance unique amongst the endonucleases characterized so far.

Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.

Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation.

Ion pairs and their role in modulating stability of cold- and warm-active uracil DNA glycosylase.

MD simulations and continuum electrostatics calculations have been used to study the observed differences in thermostability of cold- and warm-active uracil DNA glycosylase (UDG). The present study focuses on the role of ion pairs and how they affect the thermal stability of the two enzymes. Analysis of the MD generated structural ensembles show that cod UDG (cUDG) and human UDG (hUDG) have 11 and 12 ion pairs which are present in at least 30% of the conformations. The electrostatic contribution of the ion pairs, computed using continuum electrostatics, is slightly more favorable in cUDG at 298 K. This is primarily attributed to more optimized interactions between the ion pairs and nearby dipoles/charges in cUDG. More global salt bridges are found in hUDG and are more stabilizing when compared to cUDG, possibly playing a role in maintaining overall stability and reducing conformational entropy. Both enzymes contain one three-member ionic network, but the one found in hUDG is far more stabilizing. Our results also suggest that care should be taken when performing statistical analysis of crystal structures with respect to ion pairs, and that crystallization conditions must be carefully examined when performing such analysis.

Sequence comparison and environmental adaptation of a bacterial endonuclease.

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.