Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen

In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.     

FOLy: an integrated database for the classification and functional annotation of fungal oxidoreductases potentially involved in the degradation of lignin and related aromatic compounds

The breakdown of lignin by fungi is a key step during carbon recycling in terrestrial ecosystems. This process is of great interest for green and white biotechnological applications. Given the importance of these enzymatic processes, we have classified the enzymes potentially involved in lignin catabolism into sequence-based families and integrated them in a newly developed database, designated Fungal Oxidative Lignin enzymes (FOLy). Families were defined after sequence similarity searches starting from protein sequences and validated by the convergence of results with biochemical experiments reported in the literature. The resulting database was applied as a tool for the functional annotation of genomes from different fungi, namely (i) the Basidiomycota Coprinopsis cinerea, Phanerochaete chrysosporium and Ustilago maydis and (ii) the Ascomycota Aspergillus nidulans and Trichoderma reesei. Genomic comparison of the oxidoreductases of these fungi revealed significant differences in the putative enzyme arsenals. Two Ascomycota fungal genomes were annotated and new candidate genes were identified that could be useful for lignin degradation and (or) melanin synthesis, and their function investigated experimentally. This database efforts aims at providing the means to get new insights for the understanding and biotechnological exploitation of the lignin degradation. A WWW server giving access to the routinely updated FOLy classifications of enzymes potentially involved in lignin degradation can be found at http://foly.esil.univ-mrs.fr.     

Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A

The three-dimensional structure of Aspergillus aculeatus beta-1,4-galactanase (AAGAL), an enzyme involved in pectin degradation, has been determined by multiple isomorphous replacement to 2.3 and 1.8 A resolution at 293 and 100 K, respectively. It represents the first known structure for a polysaccharidase with this specificity and for a member of glycoside hydrolase family 53 (GH-53). The enzyme folds into a (beta/alpha)(8) barrel with the active site cleft located at the C-terminal side of the barrel consistent with the classification of GH-53 in clan GH-A, a superfamily of enzymes with common fold and catalytic machinery but diverse specificities. Putative substrate-enzyme interactions were elucidated by modeling of beta-1,4-linked galactobioses into the possible substrate binding subsites. The structure and modeling studies identified five potential subsites for the binding of galactans, of which one is a pocket suited for accommodating the arabinan side chain in arabinogalactan, one of the natural substrates. A comparison with the substrate binding grooves of other Clan GH-A enzymes suggests that shape complementarity is crucial in determining the specificity of polysaccharidases.     

Evolutionary and mechanistic relationships between glycosidases acting on alpha- and beta-bonds

Because of the fast accumulation of sequences derived from genome sequencing efforts, the sampling of the sequence space in glycosidase and related enzyme families is such that sensitive sequence similarity detection methods like PSI-BLAST are now able to reveal distant, but clear, structural and evolutionary relations between glycosidases acting on alpha- and beta-bonds. We have observed this trend within groups of glycosidases with completely different folds. We postulate that the evolutionary interconversion between alpha- and beta-acting glycosidases was greatly facilitated by the fact that both types share a similar axial orientation of the glycosidic bond in the reactive bound substrate. Glycosides in the beta anomeric configuration, require a sugar ring distortion, resulting in an axial orientation of the glycosidic bond, equivalent to that of an alpha glycosidic bond, prior to displacement by nucleophilic substitution.     

Structural determinants of substrate specificity in family 1 beta-glucosidases: novel insights from the crystal structure of sorghum dhurrinase-1, a plant beta-glucosidase with strict specificity, in complex with its natural substrate

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.     

A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana

The synthesis, modification, and breakdown of carbohydrates is one of the most fundamentally important reactions in nature. The structural and functional diversity of glycosides is mirrored by a vast array of enzymes involved in their synthesis (glycosyltransferases), modification (carbohydrate esterases) and breakdown (glycoside hydrolases and polysaccharide lyases). The importance of these processes is reflected in the dedication of 1-2% of an organism's genes to glycoside hydrolases and glycosyltransferases alone. In plants, these processes are of particular importance for cell-wall synthesis and expansion. starch metabolism, defence against pathogens, symbiosis and signalling. Here we present an analysis of over 730 open reading frames representing the two main classes of carbohydrate-active enzymes, glycoside hydrolases and glycosyltransferases, in the genome of Arabidopsis thaliana. The vast importance of these enzymes in cell-wall formation and degradation is revealed along with the unexpected dominance of pectin degradation in Arabidopsis, with at least 170 open-reading frames dedicated solely to this task.     

Nuclear genome sequence of the plastid-lacking cryptomonad <Emphasis Type="Italic">Goniomonas avonlea</Emphasis> provides insights into the evolution of secondary plastids

Background

The evolution of photosynthesis has been a major driver in eukaryotic diversification. Eukaryotes have acquired plastids (chloroplasts) either directly via the engulfment and integration of a photosynthetic cyanobacterium (primary endosymbiosis) or indirectly by engulfing a photosynthetic eukaryote (secondary or tertiary endosymbiosis). The timing and frequency of secondary endosymbiosis during eukaryotic evolution is currently unclear but may be resolved in part by studying cryptomonads, a group of single-celled eukaryotes comprised of both photosynthetic and non-photosynthetic species. While cryptomonads such as Guillardia theta harbor a red algal-derived plastid of secondary endosymbiotic origin, members of the sister group Goniomonadea lack plastids. Here, we present the genome of Goniomonas avonlea—the first for any goniomonad—to address whether Goniomonadea are ancestrally non-photosynthetic or whether they lost a plastid secondarily.

Results

We sequenced the nuclear and mitochondrial genomes of Goniomonas avonlea and carried out a comparative analysis of Go. avonlea, Gu. theta, and other cryptomonads. The Go. avonlea genome assembly is ~?92 Mbp in size, with 33,470 predicted protein-coding genes. Interestingly, some metabolic pathways (e.g., fatty acid biosynthesis) predicted to occur in the plastid and periplastidal compartment of Gu. theta appear to operate in the cytoplasm of Go. avonlea, suggesting that metabolic redundancies were generated during the course of secondary plastid integration. Other cytosolic pathways found in Go. avonlea are not found in Gu. theta, suggesting secondary loss in Gu. theta and other plastid-bearing cryptomonads. Phylogenetic analyses revealed no evidence for algal endosymbiont-derived genes in the Go. avonlea genome. Phylogenomic analyses point to a specific relationship between Cryptista (to which cryptomonads belong) and Archaeplastida.

Conclusion

We found no convincing genomic or phylogenomic evidence that Go. avonlea evolved from a secondary red algal plastid-bearing ancestor, consistent with goniomonads being ancestrally non-photosynthetic eukaryotes. The Go. avonlea genome sheds light on the physiology of heterotrophic cryptomonads and serves as an important reference point for studying the metabolic “rewiring” that took place during secondary plastid integration in the ancestor of modern-day Cryptophyceae.