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.     

Functional Analysis of Family GH36 α-Galactosidases from Ruminococcus gnavus E1: Insights into the Metabolism of a Plant Oligosaccharide by a Human Gut Symbiont

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14–21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex]₁₂ and [Hex]₈, respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.     

Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

Background

The enzymatic hydrolysis of α−mannosides is catalyzed by glycoside hydrolases (GH), termed α−mannosidases. These enzymes are found in different GH sequence–based families. Considerable research has probed the role of higher eukaryotic “GH38” α−mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 α−mannosidase II, which has been shown to be a retaining α−mannosidase that targets both α−1,3 and α−1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)₅(GlcNAc)₂ hybrid N-glycans to GlcNAc(Man)₃(GlcNAc)₂. Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 α−mannosidases whose activity and specificity is unknown.

Methodology/Principal Findings

Here we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an α−mannosidase with specificity for α−1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 Å resolution and in complex with the inhibitor swainsonine (K _i 18 µM) at 2.6 Å, reveals a canonical GH38 five-domain structure in which the catalytic “–1” subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn²⁺ ion. In contrast, the “leaving group” subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity.

Conclusions/Significance

Although the in vivo function of this streptococcal GH38 α−mannosidase remains unknown, it is shown to be an α−mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Evolutionary History of Eukaryotic α-Glucosidases from the α-Amylase Family

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.     

The Tetramer Structure of the Glycoside Hydrolase Family 27 α-Galactosidase I from Umbelopsis vinacea

The crystal structure of Umbelopsis vinacea α-galactosidase I, which belongs to glycoside hydrolase family 27, was determined at 2.0 Å resolution. The monomer structure was well conserved with those of glycoside hydrolase family 27 enzymes. The biological tetramer structure of this enzyme was constructed by the crystallographic 4-fold symmetry, and tetramerization appeared to be caused by three inserted peptides that were involved in the tetramer interface. The quaternary structure indicated that the substrate specificity of this enzyme might be related to the tetramer formation. Three N-glycosylated sugar chains were observed, and their structures were found to be of the high-mannose type.     

Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3 l-Araf substituents from disubstituted Xylp residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l-Araf substituents, confirming the ability of SthAbf62A to remove α-l-Araf residues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylp sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l-arabinose binding pocket.     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

Sequence-Structural Features and Evolutionary Relationships of Family GH57 α-Amylases and Their Putative α-Amylase-Like Homologues

The glycoside hydrolase family 57 (GH57) contains α-amylase and a few other amylolytic specificities. It counts ~400 members from Archaea (1/4) and Bacteria (3/4), mostly of extremophilic prokaryotes. Only 17 GH57 enzymes have been biochemically characterized. The main goal of the present bioinformatics study was to analyze sequences having the clear GH57 α-amylase features. Of the 107 GH57 sequences, 59 were evaluated as α-amylases (containing both GH57 catalytic residues), whereas 48 were assigned as GH57 α-amylase-like proteins (having a substitution in one or both catalytic residues). Forty-eight of 59 α-amylases were from Archaea, but 42 of 48 α-amylase-like proteins were of bacterial origin. The catalytic residues were substituted in most cases in Bacteroides and Prevotella by serine (instead of catalytic nucleophile glutamate) and glutamate (instead of proton donor aspartate). The GH57 α-amylase specificity has thus been evolved and kept enzymatically active mainly in Archaea.     

Phylogenetic Analysis: How Old are the Parts of Your Body?

According to the National Academy of Sciences, biology students in the USA are not being adequately prepared for successful futures. Of paramount concern is a lack of sufficient training in quantitative and computational skills, which are needed to compete effectively for an array of educational and occupational opportunities. In this paper, we introduce a classroom exercise that invites students to solve a simple biological problem and illustrates the need for a computer-assisted strategy to arrive at a solution. The exercise invites students to consider the question “How old are the parts of your body?” Some features of the human body are more ancient than others. For example, our bodies have both hair and backbones, but backbones arose much earlier in evolutionary history. Our exercise relies upon MEGA 4.0, a free, visually appealing, and intuitive computer program that allows students to gather DNA or protein sequences from electronic databases, then use them to infer phylogenetic trees. Student-inferred phylogenies are used to explore the relative order in which diverse aspects of the human form evolved. In the process, students are trained to use powerful features of MEGA and encouraged through group discussion to consider additional applications of the technology they have learned. Our lesson plan includes a brief video, a web site with essential terminology and links for further exploration, a hands-on experience using MEGA, and a follow-up discussion.     

Phylogenomic Analysis of the α Proteasome Gene Family from Early-Diverging Eukaryotes

We employed a phylogenomic approach to study the evolution of α subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven α proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple α subunit sequences generated single α proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct α proteasome genes each. The presence of seven distinct α proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the α proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the α proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented. Received: 14 February 2000 / Accepted: 14 August 2000     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Molecular Evolution of the Primate α-/θ-Defensin Multigene Family

The primate α-/θ-defensin multigene family encodes versatile endogenous cationic and amphipathic peptides that have broad-spectrum antibacterial, antifungal and antiviral activity. Although previous studies have reported that α-/θ-defensin (DEFA/DEFT) genes are under birth-and-death evolution with frequent duplication and rapid evolution, the phylogenetic relationships of the primate DEFA/DEFT genes; the genetic bases for the existence of similar antimicrobial spectra among closely related species; and the evolutionary processes involved in the emergence of cyclic θ-defensins in Old World monkeys and their subsequent loss of function in humans, chimpanzees and gorillas require further investigation. In this study, the DEFA/DEFT gene repertoires from primate and treeshrew were collected, followed by detailed phylogenetic, sequence and structure, selection pressure and comparative genomics analyses. All treeshrew, prosimian and simian DEFA/DEFT genes are grouped into two major clades, which are tissue-specific for enteric and myeloid defensins in simians. The simian enteric and myeloid α-defensins are classified into six functional gene clusters with diverged sequences, variable structures, altered functional constraints and different selection pressures, which likely reflect the antimicrobial spectra among closely related species. Species-specific duplication or pseudogenization within each simian cluster implies that the antimicrobial spectrum is ever-shifting, most likely challenged by the ever-changing pathogen environment. The DEFT evolved from the myeloid DEFA8. The prosegment of θ-defensin is detected with adaptive changes coevolving with the new protein fold of mature peptide, coincident with the importance of the prosegment for the correct folding of the mature peptide. Lastly, a less-is-hitchhiking hypothesis was proposed as a possible explanation for the expansion of pseudogene DEFTP and the loss of functional DEFT, where the gain or loss of the hitchhiker is determined by its adjacent driver gene during the birth-and-death evolutionary process.     

Phylogenetic Origin of the MAL and IMA α-Glucosidases of the International Genetic Line of Saccharomyces cerevisiae S288C

Microbiology - Taking into account the accepted concept of the ancient whole genome duplication (WGD) in the yeast genus Saccharomyces, comparative analysis of the multiple α-glucosidases MAL...     

In silico identification of catalytic residues and domain fold of the family GH119 sharing the catalytic machinery with the α-amylase family GH57

The glycoside hydrolase family 119 (GH119) contains the α-amylase from Bacillus circulans and five other hypothetical proteins. Until now, nothing has been reported on the catalytic residues and catalytic-domain fold of GH119. Based on a detailed in silico analysis involving sequence comparison in combination with BLAST searches and structural modelling, an unambiguous relationship was revealed between the families GH119 and GH57. This includes sharing the catalytic residues, i.e. Glu231 and Asp373 as catalytic nucleophile and proton donor, respectively, in the predicted catalytic (β/α)₇-barrel domain of GH119 B. circulans α-amylase. The GH57 and GH119 families may thus define a new CAZy clan.     

A Cytogenetic Analysis of the Chromosomal Region Surrounding the α-Glycerophosphate Dehydrogenase Locus of DROSOPHILA MELANOGASTER

The chromosomal region surrounding the structural gene for α-glycerophosphate dehydrogenase (αGpdh, 2-20.5) of Drosophila melanogaster has been studied in detail. Forty-three EMS-induced recessive lethal mutations and five previously identified visible mutations have been localized within the 25A-27D region of chromosome 2 by deficiency mapping and in some cases by a recombination analysis. The 43 lethal mutations specify 17 lethal loci. αGpdh has been localized to a single polytene chromosome band, 25F5, and there apparently are no lethals that map to the αGpdh locus.     

Inverting character of family GH115 α-glucuronidases

α-Glucuronidases of glycoside hydrolase family 115 of the xylose-fermenting yeast Pichia stipitis and wood-destroying fungus Schizophyllum commune liberate 4-O-methyl-d-glucuronic acid residues from aldouronic acids and glucuronoxylan. The specific activities of both enzymes depended on polymerization degree of the acidic xylooligosaccharides and were inhibited by linear β-1,4-xylooligosaccharides. These results suggest interaction of the enzyme with several xylopyranosyl residues of the xylan main chain. Using ¹H NMR spectroscopy and reduced aldopentaouronic acid (MeGlcA³Xyl₄-ol) as a substrate, it was found that both enzymes are inverting glycoside hydrolases releasing 4-O-methyl-d-glucuronic acid (MeGlcA) as its β-anomer.     

Structure–Function Analysis of the ADAM Family of Disintegrin-Like and Metalloproteinase-Containing Proteins (Review)

The ADAMs belong to a disintegrin-like and metalloproteinase-containing protein family that are zinc-dependent metalloproteinases. These proteins share all or some of the following domain structure: a signal peptide, a propeptide, a metalloproteinase, a disintegrin, a cysteine-rich, and an epidermal growth factor (EGF)-like domains, a transmembrane region, and a cytoplasmic tail. ADAMs are widely distributed in many organs, tissues, and cells, such as brain, testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone, and muscle. These proteins are capable of four potential functions: proteolysis, adhesion, fusion, and intracellular signaling. Because the number of ADAM genes has grown rapidly and the biological functions of most members are unclear, this review analyzes the protein structures and functions, their activation and processing, their known and potential activities, and their evolutionary relationships. A sequence alignment of human ADAMs is compiled and their homology and physical data are calculated. The conceivable functions of ADAMs in reproduction, development, and diseases are also discussed.     

Phylogenetic Relationship ofPopulusSections by ITS Sequence Analysis

ITS sequences of 15 representative species of five sections in the genus Populus L. were determined. By using direct sequencing of PCR product, it was found that the fragments of internal transcribed spacers (ITS) are about 594 bp in length. The length of ITS-1 and ITS-2 is about 220 bp and 210 bp, respectively, while that of 5.8s is 164 bp. Its G+C content is about 69.0%. The number of phylogenetically informative loci is higher in ITS-2 than in ITS-1. Transversion and transition are two main factors that drive the ITS evolution, and more insertions and deletions occurred in ITS-2. Taking Salix matsudana Koidz. and Salix suchowensis Cheng as outgroups, phylogenetic analysis of ITS sequences using PAUP 4.0 software indicated that Populus is monophyletic group and can be divided into two main clades. One is the section Leuce , and the other is the remaining sections.