Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara.

Background: Cellulases play a key role in the enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. Thus, the discovery of novel cellulases from exotic biological niches is of great interest as they may present properties that are valuable in the biorefining of lignocellulosic biomass.

Results: We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multi-domain enzyme from the unusual gill endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara.

Intrinsic Strand-Incision Activity of Human UNG: Implications for Nick Generation in Immunoglobulin Gene Diversification.

Uracil arises in cellular DNA by cytosine (C) deamination and erroneous replicative incorporation of deoxyuridine monophosphate opposite adenine. The former generates C → thymine transition mutations if uracil is not removed by uracil-DNA glycosylase (UDG) and replaced by C by the base excision repair (BER) pathway. The primary human UDG is hUNG.

Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling.

Iron is an essential nutrient for bacteria, however its propensity to form toxic hydroxyl radicals at high intracellular concentrations, requires its acquisition to be tightly regulated. Ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as a transcriptional regulator in maintaining iron metabolism in bacteria and is a highly interesting target in the design of new antibacterial drugs. Fur mutants have been shown to exhibit decreased virulence in infection models.

The Gene Is Activated to Alleviate Mutagenesis by an Oxidized Deoxynucleoside.

The cellular methyl donor -adenosylmethionine (SAM) and other endo/exogenous agents methylate DNA bases non-enzymatically into products interfering with replication and transcription. An important product is 3-methyladenine (mA), which in is removed by mA-DNA glycosylase I (Tag) and II (AlkA). The gene is constitutively expressed, while is induced by sub-lethal concentrations of methylating agents.

The crystal structure of the tetrameric DABA-aminotransferase EctB, a rate-limiting enzyme in the ectoine biosynthesis pathway.

l-2,4-diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω-position of substrates, and is the intial and rate-limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.

Characterization of an intertidal zone metagenome oligoribonuclease and the role of the intermolecular disulfide bond for homodimer formation and nuclease activity.

The gene encoding MG Orn has been identified from a metagenomic library created from the intertidal zone in Svalbard and encodes a protein of 184 amino acid residues. The mg orn gene has been cloned, recombinantly expressed in Escherichia coli, and purified to homogeneity. Biochemical characterization of the enzyme showed that it efficiently degrades short RNA oligonucleotide substrates of 2mer to 10mer of length and has an absolute requirement for divalent cations for optimal activity.

Temperature adaptation of DNA ligases from psychrophilic organisms.

DNA ligases operating at low temperatures have potential advantages for use in biotechnological applications. For this reason, we have characterized the temperature optima and thermal stabilities of three minimal Lig E-type ATP-dependent DNA ligase originating from Gram-negative obligate psychrophilic bacteria. The three ligases, denoted Vib-Lig, Psy-Lig, and Par-Lig, show a remarkable range of thermal stabilities and optima, with the first bearing all the hallmarks of a genuinely cold-adapted enzyme, while the latter two have activity and stability profiles more typical of mesophilic proteins.

Mutational analysis of the pro-peptide of a marine intracellular subtilisin protease supports its role in inhibition.

Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog.

Biochemical characterization of ParI, an orphan C5-DNA methyltransferase from Psychrobacter arcticus 273-4.

Cytosine-specific DNA methyltransferases are important enzymes in most living organisms. In prokaryotes, most DNA methyltransferases are members of the type II restriction-modification system where they methylate host DNA, thereby protecting it from digestion by the accompanying restriction endonucleases. DNA methyltransferases can also act as solitary enzymes having important roles in controlling gene expression, DNA replication, cell cycle and DNA post-replicative mismatch repair.

Discovery and characterization of a thermostable two-domain GH6 endoglucanase from a compost metagenome.

Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines.

Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å.

Properties and distribution of a metallo-β-lactamase (ALI-1) from the fish pathogen Aliivibrio salmonicida LFI1238.

Objectives: To characterize the chromosome-encoded metallo-β-lactamase (MBL) from the psychrophilic, marine fish-pathogenic bacterium Aliivibrio salmonicida LFI1238 and check for the presence of the gene in other Aliivibrio isolates both connected to the fish-farming industry and from the environment.

Methods: The MBL gene was cloned and intracellularly expressed in Escherichia coli. Kinetic parameters, NaCl dependence, pH optimum and temperature optimum were determined using purified enzyme.

His224 alters the R2 drug binding site and Phe218 influences the catalytic efficiency of the metallo-β-lactamase VIM-7.

Metallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments.

Structure-function studies of an unusual 3-methyladenine DNA glycosylase II (AlkA) from Deinococcus radiodurans.

3-Methyladenine DNA glycosylase II (AlkA) is a DNA-repair enzyme that removes alkylated bases in DNA via the base-excision repair (BER) pathway. The enzyme belongs to the helix-hairpin-helix (HhH) superfamily of DNA glycosylases and possesses broad substrate specificity. In the genome of Deinococcus radiodurans, two genes encoding putative AlkA have been identified (Dr_2074 and Dr_2584).

The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism.

Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35 Å and a quaternary complex of CtIDH with NADP(+), isocitrate and Mg(2+) to 2.

Structural and computational investigations of VIM-7: insights into the substrate specificity of vim metallo-β-lactamases.

The presence of metallo-β-lactamases (MBLs) in many clinically important human bacterial pathogens limits treatment options, as these enzymes efficiently hydrolyze nearly all β-lactam antibiotics. VIM enzymes are among the most widely distributed MBLs, but many of the individual VIM subtypes remain poorly characterized. Pseudomonas aeruginosa VIM-7 is the most divergent among VIM-type MBLs in terms of amino acid sequence.

The hyperthermophilic euryarchaeon Archaeoglobus fulgidus repairs uracil by single-nucleotide replacement.

Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transition mutations if uracil is not repaired by the DNA base excision repair (BER) pathway. Since deamination increases rapidly with temperature, hyperthermophiles, in particular, are expected to succumb to such damage. There has been only one report of crenarchaeotic BER showing strong similarities to that in most eukaryotes and bacteria for hyperthermophilic Archaea.

Experimental and computational characterization of the ferric uptake regulator from Aliivibrio salmonicida (Vibrio salmonicida).

The Ferric uptake regulator (Fur) is a global transcription factor that affects expression of bacterial genes in an iron-dependent fashion. Although the Fur protein and its iron-responsive regulon are well studied, there are still important questions that remain to be answered. For example, the consensus Fur binding site also known as the "Fur box" is under debate, and it is still unclear which Fur residues directly interact with the DNA.

Allosteric communication between cAMP binding sites in the RI subunit of protein kinase A revealed by NMR.

The activation of protein kinase A involves the synergistic binding of cAMP to two cAMP binding sites on the inhibitory R subunit, causing release of the C subunit, which subsequently can carry out catalysis. We used NMR to structurally characterize in solution the RIalpha-(98-381) subunit, a construct comprising both cyclic nucleotide binding (CNB) domains, in the presence and absence of cAMP, and map the effects of cAMP binding at single residue resolution. Several conformationally disordered regions in free RIalpha become structured upon cAMP binding, including the interdomain alphaC:A and alphaC':A helices that connect CNB domains A and B and are primary recognition sites for the C subunit.

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.

Opposite-base dependent excision of 5-formyluracil from DNA by hSMUG1.

Purpose: The aim of this study was to determine the excision efficiency of hSMUG1 (human single-strand-selective monofunctional uracil-DNA glycosylase) for 5-formyluracil (fU), a major thymine lesion formed by ionizing radiation, opposite all normal bases in DNA, to possibly explain mutation induction by fU in the DNA of mammalian cells.

Materials And Methods: An enzymatically [(32)P]labelled fU-containing 36 nucleotide DNA sequence plus its complementary sequence (with an A, C, G or T residue inserted opposite fU) was subjected to hSMUG1 in a pH 7.5-buffer, followed by NaOH-mediated cleavage of the resultant abasic sites.

The first structure of a cold-adapted superoxide dismutase (SOD): biochemical and structural characterization of iron SOD from Aliivibrio salmonicida.

Superoxide dismutases (SODs) are metalloenzymes that catalyse the dismutation of the superoxide radical anion into O(2) and H(2)O(2) in a two-step reaction. The crystal structure of the iron superoxide dismutase from the cold-adapted and fish-pathogenic bacterium Aliivibrio salmonicida (asFeSOD) has been determined and refined to 1.7 A resolution.

Structure of Deinococcus radiodurans tunicamycin-resistance protein (TmrD), a phosphotransferase.

The open-reading frame (ORF) DR_1419 in the Deinococcus radiodurans genome is annotated as a representative of the wide family of tunicamycin-resistance proteins as identified in a range of bacterial genomes. The D. radiodurans ORF DR_1419 was cloned and expressed; the protein TmrD was crystallized and its X-ray crystal structure was determined to 1.

Crystal structure and mutational study of RecOR provide insight into its mode of DNA binding.

The crystal structure of the complex formed between Deinococcus radiodurans RecR and RecO (drRecOR) has been determined. In accordance with previous biochemical characterisation, the drRecOR complex displays a RecR:RecO molecular ratio of 2:1. The biologically relevant drRecOR entity consists of a heterohexamer in the form of two drRecO molecules positioned on either side of the tetrameric ring of drRecR, with their OB (oligonucleotide/oligosaccharide-binding) domains pointing towards the interior of the ring.

Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability.

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). CpPAH displays highest activity with tetrahydrobiopterin (BH(4)) as cofactor and at 25 degrees C (15 degrees C above the optimal growth temperature).