Ustiloxin biosynthetic machinery is not compatible between Aspergillus flavus and Ustilaginoidea virens.

Ustiloxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) first reported in Ascomycetes. Originally identified as metabolites of the rice pathogenic fungus Ustilaginoidea virens, they were recently identified among the metabolites of the mold Aspergillus flavus, along with their corresponding biosynthetic gene cluster. Ustilaginoidea virens produces ustiloxins A and B, whereas A.

Improvement of ST0452 -Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering.

We showed previously that the Y97N mutant of the ST0452 protein, isolated from , exhibited over 4 times higher -acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of -acetylglucosamine (GlcNAc) in the complex without metal ions.

Identification of a Direct Biosynthetic Pathway for UDP--Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii.

Most organisms, from to , synthesize UDP--acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP--acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In , the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon , predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon.

Comparison of the microbial community structure between inflamed and non-inflamed sites in patients with ulcerative colitis.

Background And Aim: The gut microbiota is suggested to play an important role in the pathogenesis of ulcerative colitis (UC). However, interindividual and spatial variations hamper the identification of UC-related changes. We thus investigated paired mucosa-associated microbiota obtained from both inflamed and non-inflamed sites of UC patients and corresponding sites of non-inflammatory bowel disease (IBD) controls.

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

Unlabelled: The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant.

Current Technological Improvements in Enzymes toward Their Biotechnological Applications.

Enzymes from extremophiles are creating interest among researchers due to their unique properties and the enormous power of catalysis at extreme conditions. Since community demands are getting more intensified, therefore, researchers are applying various approaches viz. metagenomics to increase the database of extremophilic species.

Moss Chloroplasts Are Surrounded by a Peptidoglycan Wall Containing D-Amino Acids.

It is believed that the plastids in green plants lost peptidoglycan (i.e., a bacterial cell wall-containing d-amino acids) during their evolution from an endosymbiotic cyanobacterium.

Mutation of the gene encoding the ribonuclease P RNA in the hyperthermophilic archaeon Thermococcus kodakarensis causes decreased growth rate and impaired processing of tRNA precursors.

Ribonuclease P (RNase P) catalyzes the processing of 5' leader sequences of tRNA precursors in all three phylogenetic domains. RNase P also plays an essential role in non-tRNA biogenesis in bacterial and eukaryotic cells. For archaeal RNase Ps, additional functions, however, remain poorly understood.

A longer finger-subdomain of family A DNA polymerases found by metagenomic analysis strengthens DNA binding and primer extension abilities.

The family A DNA polymerases from thermophilic bacteria are useful for PCR. The DNA polymerase from Thermus aquaticus (Taq polymerase) was the original enzyme used when practical PCR was developed, and it has remained the standard enzyme for PCR to date. Knowledge gained from structure-function relationship studies of Taq polymerase is applicable to create PCR enzymes with enhanced performance.

Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3.

Mannosylglycerate is known as a compatible solute, and plays important roles for salinity adaptation and high temperature stability of microorganisms. In the gene cluster for the mannosylglycerate biosynthetic pathway predicted from the genomic data of Pyrococcus horikoshii OT3, the PH0925 protein was found as a putative bifunctional enzyme with phosphomannose isomerase (PMI) and mannose-1-phosphate guanylyltransferase (Man-1-P GTase) activities, which can synthesize GDP-mannose when accompanied by a phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme (PH0923). The recombinant PH0925 protein, expressed in E.

Crystal Structures of a Hyperthermophilic Archaeal Homoserine Dehydrogenase Suggest a Novel Cofactor Binding Mode for Oxidoreductases.

NAD(P)-dependent dehydrogenases differ according to their coenzyme preference: some prefer NAD, others NADP, and still others exhibit dual cofactor specificity. The structure of a newly identified archaeal homoserine dehydrogenase showed this enzyme to have a strong preference for NADP. However, NADP did not act as a cofactor with this enzyme, but as a strong inhibitor of NAD-dependent homoserine oxidation.

Experimental confirmation of a whole set of tRNA molecules in two archaeal species.

Based on the genomic sequences for most archaeal species, only one tRNA gene (isodecoder) is predicted for each triplet codon. This observation promotes analysis of a whole set of tRNA molecules and actual splicing patterns of interrupted tRNA in one organism. The entire genomic sequences of two Creanarchaeota, Aeropyrum pernix and Sulfolobus tokodaii, were determined approximately 15 years ago.

Characterization of the amino acid residues mediating the unique amino-sugar-1-phosphate acetyltransferase activity of the archaeal ST0452 protein.

The ST0452 protein from the thermophilic archaean Sulfolobus tokodaii has been identified as an enzyme with multiple sugar-1-phosphate nucleotidylyltransferase and amino-sugar-1-phosphate acetyltransferase (amino-sugar-1-P AcTase) activities. Analysis of the protein showed that in addition to glucosamine-1-phosphate (GlcN-1-P) AcTase activity, it possesses unique galactosamine-1-phosphate (GalN-1-P) AcTase activity not detected in any other proteins. Comparison of the crystal structures of the ST0452 protein and GlmU from Escherichia coli (EcGlmU), which possesses only GlcN-1-P AcTase activity, showed that the overall sequence identity between these two proteins is less than 25 %, but the amino acid residues predicted to comprise the catalytic center of EcGlmU are conserved in the ST0452 protein.

Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.

DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. Thermostable DNA polymerases are especially useful and became quite valuable after the development of PCR technology. A DNA polymerase from Thermus aquaticus (Taq polymerase) is the most famous DNA polymerase as a PCR enzyme, and has been widely used all over the world.

The structure of hyperthermophilic β-N-acetylglucosaminidase reveals a novel dimer architecture associated with the active site.

Unlabelled: The β-N-acetylglucosaminidase from the hyperthermophilic bacteria Thermotoga maritima (NagA) hydrolyzes chitooligomers into monomer β-N-acetylglucosamine. Although NagA contains a highly conserved sequence motif found in glycoside hydrolase (GH) family 3, it can be distinguished from other GH family 3 β-N-acetylglucosaminidases by its substrate specificity and biological assembly. To investigate its unique structure around the active site, we determined the crystal structure of NagA at a resolution of 2.

Cloning, expression and characteristics of a novel alkalistable and thermostable xylanase encoding gene (Mxyl) retrieved from compost-soil metagenome.

Background: The alkalistable and thermostable xylanases are in high demand for pulp bleaching in paper industry and generating xylooligosaccharides by hydrolyzing xylan component of agro-residues. The compost-soil samples, one of the hot environments, are expected to be a rich source of microbes with thermostable enzymes.

Methodology/principal Findings: Metagenomic DNA from hot environmental samples could be a rich source of novel biocatalysts.

Comparative genome analysis between Aspergillus oryzae strains reveals close relationship between sites of mutation localization and regions of highly divergent genes among Aspergillus species.

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A.

Creation of a thermostable NADP⁺-dependent D-amino acid dehydrogenase from Ureibacillus thermosphaericus strain A1 meso-diaminopimelate dehydrogenase by site-directed mutagenesis.

A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH.

Acidophilic bacteria and archaea: acid stable biocatalysts and their potential applications.

Acidophiles are ecologically and economically important group of microorganisms, which thrive in acidic natural (solfataric fields, sulfuric pools) as well as artificial man-made (areas associated with human activities such as mining of coal and metal ores) environments. They possess networked cellular adaptations to regulate pH inside the cell. Several extracellular enzymes from acidophiles are known to be functional at much lower pH than the cytoplasmic pH.

Identification of an entire set of tRNA molecules and characterization of cleavage sites of the intron-containing tRNA precursors in acidothermophilic crenarchaeon Sulfolobus tokodaii strain7.

The acidothermophilic crenarchaeon, Sulfolobus tokodaii strain7, was isolated from a hot spring in Beppu, Kyushu, Japan. Whole genomic data of this microorganism indicated that among 46 putative tRNA genes identified, 24 were interrupted tRNA genes containing an intron. A sequence comparison between the cDNA sequences for unspliced and spliced tRNAs indicated that all predicted tRNAs were expressed and all intron portions were spliced in this microorganism.

Structure of flap endonuclease 1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5'-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes.

Artificial self-sufficient P450 in reversed micelles.

Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents.

Identification of novel acetyltransferase activity on the thermostable protein ST0452 from Sulfolobus tokodaii strain 7.

A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein.

Expression and functional analysis of a predicted AtsG arylsulphatase identified from Mycobacterium tuberculosis genomic data.

Sulphatase family enzymes hydrolyse the sulphate ester, found on the pathogens cell surface and playing an important role for host-pathogen interaction. The AtsG, homologue of arylsulphatase, predicted in the Mycobacterium tuberculosis genomic data, was successfully expressed in Escherichia coli. The recombinant AtsG protein exhibited hydrolysis of para-nitrophenyl sulphate and para-nitrocatechol sulphate, and binding affinity to the heparin-sepharose resin.