Phylogenetic Analysis of the Plant Endo-β-1,4-Glucanase Gene Family

Phylogenetic analysis of the endo--1,4-glucanase gene family of Arabidopsis and other plants revealed a clear distinction in three subfamilies (, , and ). The - and -subfamily contains proteins believed to be involved in a number of physiological roles such as elongation, ripening, and abscission. The -subfamily is composed of proteins that are predicted to have a membrane-spanning domain and to be localized at the plasma membrane. Some of these proteins have been linked to cellulose biosynthesis by serving to hydrolyze a lipid-linked intermediate that acts as a primer for the elongation of -glucan chains during cellulose synthesis at the plasma membrane. Similar glucanases are important in cellulose biosynthesis in bacteria. Searches in the genomes of unrelated organisms that make cellulose, such as Ciona intestinalis and Dictyostelium discoideum, revealed the presence of membrane-linked endo--1,4-glucanases and it is suggested that these might also have a role in cellulose synthesis.     

Characterizing the Link between Glycosylation State and Enzymatic Activity of the Endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana

Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.     

Purification and Some Properties of an Endo-β,1,6-glucanase from Neurospora crassa

An endo-β-1,6-glucanase (E.C. 3.2.1.75) was purified from the culture filtrate of Neurospora crassa IFO-6O68 by chromatographies on CM-cellulofine, Con-A Sepharose 4B, and Sepharose Cl-6B followed by preparative affinity gel electrophoresis. The purified enzyme had an apparent molecular weight of 47,000. The pH and temperature optima for the activity were 5.0 and 50°C. The enzyme acted on β-1,6-glucan (Pustulan) and yielded a series of gentio-oligosaccharides with endo- type action, and finally, glucose and gentiobiose were produced. The enzyme was also able to act on N. crassa cell wall β-glucan, and a small amount of hydrolysis fragments were liberated without apparent change of the cell wall glucan molecules.     

Amino Acid Sequence and Stereoselective Hydrolytic Reaction of an Endo-1,4-β-glucanase from a Bacillus Strain

Bacillus sp. KSM-522 produces three different extracellular endo-l,4-β-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83–89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser²⁶ through Lys⁶³⁶) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50°C, respectively. By ¹H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl β-d-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.     

Evolutionary Genomics Reveals Lineage-Specific Gene Loss and Rapid Evolution of a Sperm-Specific Ion Channel Complex: CatSpers and CatSperβ

The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca²⁺ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperβ. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperβ, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperβ originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperβ through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca²⁺ channels and their adaptation to sperm biology through metazoan evolution.     

Cloning and Characterization of the Gene Encoding Endo-β-1,3-glucanase from Arthrobacter sp. NHB-10

The gluA gene, encoding an endo-β-1,3-glucanase from Arthrobacter sp. (strain NHB-10), was cloned and analyzed. The deduced endo-β-1,3-glucanase amino acid sequence was 750 amino acids long and contained a 42 amino acid signal peptide with a mature protein of 708 amino acids. There was no similarity to known endo-β-1,3-glucanases, but GluA was partially similar to two fungal exo-β-1,3-glucanases in glycoside hydrolase (GH) family 55. Of five possible residues for catalysis and two motifs in two β-helix heads of GH family 55, three residues and one motif were conserved in GluA, suggesting that GluA is the first bacterial endo-β-1,3-glucanase in GH family 55. Significant similarity was also found to two proteins of unknown function from Streptomyces coelicolor A3(2) and S. avermitilis.     

Cloning and Expression of an Endo-1,6-β-D-glucanase Gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-β-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-β-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-β-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.     

Distribution and Properties of Endo-β-1,4-glucanase from a Lower Termite,Coptotermes formosanus (Shiraki)

A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.

EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and K _m were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.     

Cloning and expression in Saccharomyces cerevisiae of an endo-β-glucanase gene from a thermophilic cellulolytic anaerobe

Summary The gene encoding heat-stable -glucanase from a thermophilic cellulolytic anaerobe was recloned in Saccharomyces cerevisiae. Yeast transformant expressed the heat-stable endo--glucanase and produced a level of enzyme activity similar to the Escherichia coli transformant.This work was supported in part by the Biomass Conversion Project of the Ministry of Agriculture, Forestry and Fisheries, Japan     

Phylogenetic Analysis of Vertebrate Fibrillar Collagen Locates the Position of Zebrafish α3(I) and Suggests an Evolutionary Link Between Collagen α Chains and Hox Clusters

Type I collagen in tetrapods is usually a heterotrimeric molecule composed of two 1 and one 2 chains. In some teleosts, a third chain has been identified by chromatography, suggesting that type I collagen should also exist as an 1(I)2(I)3(I) heterotrimer. We prepared, from zebrafish, three distinct cDNAs identified to be those of the collagen 1(I), 2(I), and 3(I) chains. In this study on the evolution of fibrillar collagen chains and their relationships, an exhaustive phylogenetic analysis, using vertebrate fibrillar collagen sequences, showed that each chain constitutes a monophyletic cluster. Results obtained with the newly isolated sequences of the zebrafish showed that the 3(I) chain is phylogenetically close to the 1(I) chain and support the hypothesis that the 3(I) chain arose from a duplication of the 1(I) gene. The duplication might occur during the duplication of the actinopterygian genome, soon after the divergence of actinopterygians and sarcopterygians, a hypothesis supported by the demonstration of a syntenic evolution between a set of fibrillar collagen genes and Hox clusters in mammals. An evolutionary scenario is proposed in which phylogenetic relationships of the chains of fibrillar collagens of vertebrates could be related to Hox cluster history. Present address (Laure Bonnaud): Institut Jacques Monod, Tour 43, CNRS, Universités Paris 6 et 7, Equipe Evolution du Développement des Nématodes, 2 place Jussieu, 75005 Paris, France     

Molecular Architecture of the Mn-dependent Lactonase UlaG Reveals an RNase-like Metallo-β-lactamase Fold and a Novel Quaternary Structure

The ulaG gene, located in the ula regulon, is crucial for the catabolism of l-ascorbate under anaerobic conditions and it has been proposed to encode for the putative l-ascorbate-6-P lactonase. The ulaG gene is widespread among eubacteria, including human commensal and pathogenic genera such as Escherichia, Shigella, Klebsiella and Salmonella. Here, we report the three-dimensional structures of the apoenzyme and Mn²⁺ holoenzyme of UlaG from E. coli to 2.6 Å resolution, determined using single-wavelength anomalous diffraction phasing and molecular replacement, respectively. The structures reveal a highly specialized metallo-β-lactamase-like fold derived from an ancient structural template that was involved in RNA maturation and DNA repair. This fold has a novel quaternary architecture consisting of a hexameric ring formed by a trimer of UlaG dimers. A mononuclear Mn²⁺-binding site resides at the core of the active site, which displays micromolar affinity for Mn²⁺ and a distorted trigonal bipyramidal coordination. The active site Mn²⁺ ion can be replaced by Co²⁺ or Zn²⁺, but not by Fe³⁺. We further show that the Mn²⁺ or Co²⁺-loaded enzyme exhibits lactonase activity towards l-ascorbate 6-P, thereby providing the first direct evidence of its catalytic role in the l-ascorbate catabolic pathway. Guided by the structural homology, we show that UlaG is able to cleave phosphodiester linkages in cyclic nucleotides, suggesting that the conservation of the fold and of the key catalytic residues allows for the evolutionary acquisition of substrate specificity for novel but related substrates.     

In Vivo Analysis of Centromeric Proteins Reveals a Stem Cell-Specific Asymmetry and an Essential Role in Differentiated,Non-proliferating Cells

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Purification and Partial Characterization of an Endo-(1→3, 1→4)-β-glucanase from Rice,Oryza sativa L.

(1→3, 1→4)-β-Glucanase (EC 3.2.1.73), with a molecular weight of 34, 000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (1→3, 1→4)-β-glucans such as barley β-glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley β-glucan as the substrate suggested that the enzyme is an endo-type (1→3, 1→4)-β-glucanase.     

Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp. MEY-1

Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml^–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg^–1) was higher than toward carboxymethyl cellulose sodium (395 U mg^–1), which was different from other family 7 endo-β-glucanases.     

The X-ray Crystal Structure of an Arthrobacter protophormiae Endo-β-N-Acetylglucosaminidase Reveals a (β/α)8 Catalytic Domain, Two Ancillary Domains and Active Site Residues Key for Transglycosylation Activity

Glycoside hydrolase family GH85 is a family of endo-β-N-acetylglucosaminidases that is responsible for the hydrolysis of β-1,4 linkage in the N,N-diacetylchitobiose core of N-linked glycans. The endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) is of particular interest, given its increasing use for the chemoenzymatic synthesis of bespoke N-glycans using N-glycan oxazolines as glycosyl donors. The E173Q variant of Endo-A is especially attractive for synthesis, as it is hydrolytically impaired but still able to catalyze N-glycan synthesis by transglycosylation using activated oxazoline donors. Here we present the three-dimensional structure of the A. protophormiae Endo-A E173Q variant, solved by multiple-wavelength anomalous scattering methods and refined at 1.8 Å resolution. The structure reveals that GH85 enzymes display a trimodular architecture in which a (β/α)₈ catalytic domain occurs with two ancillary β-sheet modules. The active centre is fully consistent with the known neighboring-group catalytic mechanism in which E173 acts as the catalytic acid/base for reaction via an oxazoline intermediate. Of note is the presence of an asparagine in the active centre, in a position likely to interact with the acetyl NH group that, in all other known families of glycosidase using this mechanism, is an aspartate or glutamate residue. The substrate-binding surface reveals an open topography, consistent with the ability to accept a large range of glycoprotein substrates and the ability to transglycosylate other acceptors. The three-dimensional structure of this important biocatalyst reveals that residues implicated in the enhancement of transglycosylation and synthetic capacity are proximal to the active centre, where they may act to favor binding of acceptor substrates.     

Gene flow between an endangered endemic iguana, and its wide spread relative, on the island of Utila, Honduras: when is hybridization a threat?

The island endemic Ctenosaura bakeri was listed as critically endangered by the IUCN Redlist Assessment in 2004, 7 years after it was recognized as a distinct species. C. bakeri occupies a portion of Utila, a small continental island located off the northern coast of Honduras. Habitat destruction and over-harvesting are among the top threats facing this species. In addition, morphological evidence of hybridization was recently documented, raising the concern that gene flow from the common and widely distributed C. similis could threaten the genetic distinctiveness of C. bakeri. We show that hybridization occurs only at low levels and is not a current threat to C. bakeri. All ctenosaurs captured for this study were identified to species level without difficulty; none had intermediate or mosaic phenotypes. Sequence analysis of mitochondrial and nuclear markers revealed only two individuals with introgressed genotypes. Molecular analysis of the previously described hybrid showed it to be heterozygous for C. bakeri and C. similis alleles. Hybridization between these two species is possible and occurs occasionally in the wild, and the rate of hybridization could increase if habitat destruction or changes in relative abundance increase the probability of interbreeding. However, the level of gene flow indicated by current data is too low to threaten C. bakeri with genetic swamping or deleterious fitness effects.     

Cloning of Exo-β-1,3-glucanase Gene from a Marine Yeast <Emphasis Type="Italic">Williopsis saturnus</Emphasis> and Its Overexpression in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

The exo-β-1,3-glucanase structural gene (WsEXG1 gene, accession number: FJ875997.2) was isolated from both the genomic DNA and cDNA of the marine yeast Williopsis saturnus WC91-2 by inverse PCR and RT-PCR. An open reading frame of 1,254 bp encoding a 417 amino acid protein (isoelectric point: 4.5) with calculated molecular weight of 46.2 kDa was characterized. The promoter of the gene (intronless) was located from −28 to −77 and had one TATA box while its terminator contained the sequence AAGAACAATAAACAA from +1,386 to +1,401. The protein had the Family 5 glycoside hydrolase signature IGLELLNEPL and a fragment with the sequence of NLCGEWSAA, where the Glu-310 (E) was considered to be the catalytic nucleophile. The WsEXG1 gene was overexpressed in Yarrowia lipolytica Po1h and the recombinant WsEXG1 was purified and characterized. The molecular weight of the purified rWsEXG1 was 46.0 kDa. The optimal pH and temperature of the purified rWsEXG1 were 5.0°C and 40°C, respectively. The purified rWsEXG1 had high exo-β-1,3-glucanase activity. Therefore, the recombinant β-1,3-glucanase may have highly potential applications in food and pharmaceutical industries.