The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Knockout of pgdS and ggt genes improves γ‐PGA yield in B. subtilis

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ‐glutamic acid) (γ‐PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ‐PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ‐PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ‐PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ‐PGA productivity. Biotechnol. Bioeng. 2013; 110: 2006–2012. © 2013 Wiley Periodicals, Inc.     

Cloning and expression of the gene encoding <Emphasis Type="BoldItalic">Streptomyces coelicolor</Emphasis> A3(2) α-galactosidase belonging to family 36

The α-galactosidase gene of Streptomyces coelicolor A3(2) was cloned, expressed in Escherichia coli and characterized. It consisted of 1497 nucleotides encoding a protein of 499 amino acids with a predicted molecular weight of 57,385. The observed homology between the deduced amino acid sequences of the enzyme and α-galactosidase from Thermus thermophilus was over 40%. The α-galactosidase gene was assigned to family 36 of the glycosyl hydrolases. The enzyme purified from recombinant E. coli showed optimal activity at 40 °C and pH 7. The enzyme hydrolyzed p-nitrophenyl-α-D-galactopyroside, raffinose, stachyose but not melibiose and galactomanno-oligosaccharides, indicating that this enzyme recognizes not only the galactose moiety but also other substrates.     

Biosynthesis of dothistromin

Dothistromin is a mycotoxin that is remarkably similar in structure to versicolorin B, a precursor of both aflatoxin and sterigmatocystin. Dothistromin-producing fungi also produce related compounds, including some aflatoxin precursors as well as alternative forms of dothistromin. Dothistromin is synthesized by pathogenic species of Dothistroma in the red bands of pine needles associated with needle blight, but is also made in culture where it is strongly secreted into the surrounding medium. Orthologs of aflatoxin and sterigmatocystin biosynthetic genes have been found that are required for the biosynthesis of dothistromin, along with others that are speculated to be involved in the same pathway on the basis of their sequence similarity to aflatoxin genes. An epoxide hydrolase gene that has no homolog in the aflatoxin or sterigmatocystin gene clusters is also clustered with the dothistromin genes, and all these genes appear to be located on a minichromosome in Dothistroma septosporum. The dothistromin genes are expressed at an early stage of growth, suggesting a role in the first stages of plant invasion by the fungus. Future studies are expected to reveal more about the role of dothistromin in needle blight and about the genomic organization and expression of dothistromin genes: these studies will provide for interesting comparisons with these aspects of aflatoxin and sterigmatocystin biosynthesis.     

Structural and biochemical properties of lichenase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The recombinant enzyme lichenase of size 30 kDa was over-expressed using E. coli cells and purified by immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography. The enzyme displayed high activity towards lichenan and β-glucan. The enzyme showed no activity towards carboxymethyl cellulose, laminarin, galactomannan or glucomannan. Surprisingly, affinity-gel electrophoresis on native-PAGE showed that the enzyme binds only glucomannan and not lichenan or β-glucan or other manno-configured substrates. The enzyme was thermally stable between the temperatures 60°C and 70°C. Presence of Cu²⁺ ions at a concentration of 5 mM enhanced enzyme activity by 10% but higher concentrations of Cu²⁺ (>25 mM) showed a sharp fall in the enzyme activity. Heavy metal ions Ni²⁺, Co²⁺ and Zn²⁺ did not affect the activity of the enzyme at low concentrations (0–10 mM) but at higher concentrations (>10 mM), caused a decrease in the enzyme activity. The crystals of lichenase were produced and the 3-dimensional structure of native form of enzyme was previously solved at 1.50 Å. Lichenase displayed (β/α)₈-fold a common fold among many glycoside hydrolase families. A cleft was identified that represented the probable location of active site.     

N-(3,4-Dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[11C]methylphenyl)thiazol-2-yl]-1-carboxamide: A promising positron emission tomography ligand for fatty acid amide hydrolase

To visualize fatty acid amide hydrolase (FAAH) in brain in vivo, we developed a novel positron emission tomography (PET) ligand N-(3,4-dimethylisoxazol-5-yl)piperazine-4-[4-(2-fluoro-4-[¹¹C]methylphenyl)thiazol-2-yl]-1-carboxamide ([¹¹C]DFMC, [¹¹C]1). DFMC (1) was shown to have high binding affinity (IC₅₀: 6.1 nM) for FAAH. [¹¹C]1 was synthesized by C–¹¹C coupling reaction of arylboronic ester 2 with [¹¹C]methyl iodide in the presence of Pd catalyst. At the end of synthesis, [¹¹C]1 was obtained with a radiochemical yield of 20 ± 10% (based on [¹¹C]CO₂, decay-corrected, n = 5) and specific activity of 48–166 GBq/μmol. After the injection of [¹¹C]1 in mice, high uptake of radioactivity (>2% ID/g) was distributed in the lung, liver, kidney, and brain, organs with high FAAH expression. PET images of rat brains for [¹¹C]1 revealed high uptakes in the cerebellar nucleus (SUV = 2.4) and frontal cortex (SUV = 2.0), two known brain regions with high FAAH expression. Pretreatment with the FAAH-selective inhibitor URB597 reduced the brain uptake. Higher than 90% of the total radioactivity in the rat brain was irreversible at 30 min after the radioligand injection. The present results indicate that [¹¹C]1 is a promising PET ligand for imaging of FAAH in living brain.     

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.     

Isolation, heterologous expression and characterization of an endo-polygalacturonase produced by the phytopathogen Burkholderia cepacia

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Biodegradation of organophosphorus insecticides by two organophosphorus hydrolase genes (opdA and opdE) from isolated Leuconostoc mesenteroides WCP307 of kimchi origin

This study is aimed to reveal the molecular incidence of organophosphorus insecticides degradation during the fermentation of Korean food yeulmu-mulkimchi. To this end, two opdA and opdE which consist of 930 and 894 bp that encode 309 and 297 amino acids, respectively, were cloned from the Leuconostoc mesenteroides WCP307 strain that was isolated from chlorpyrifos (CP) impregnated kimchi. The Escherichia coli that harbored the opdA and opdE genes depleted a CP concentration of 72% and 83%, respectively, in an M9 medium after 6 days. The OpdA and OpdE enzymes molecular weights were estimated to be approximately 35 and 33 kDa and showed optimal activities at 30 °C with a pH of 7.0 and 6.0, respectively. However, the mutated OpdA (Ser128 → Ala128) and OpdE (Ser129 → Ala129) enzymes had no activities on OP insecticides and ρ-nitrophenyl butyrate substrates. In addition, the OpdA and OpdE enzymes showed profound catalytic activities against cadusafos, comnaphos, diazinon, dyfonate, ethoprophos, fenamiphos, methylparathion, and parathion insecticides. Therefore, it is assumed that OpdA and OpdE enzymes detoxified the pesticides contaminated kimchi composition like Chinese cabbages during fermentation. Furthermore, the OpdA and OpdE enzymes augmented the diversity of new LAB-opd enzymes group in nature.