Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes (nf2152, nf2215, nf2523, and pr2455) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, β1,2-arabinobiose (Ara2) from sugar beet arabinan (SBA), and β1,2-Ara2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both β-1,2-Ara2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications

Caractérisation fonctionnelle et structurale d'arylamine N-acétyltransférases bactériennes

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Two complementary α-fucosidases from Streptococcus pneumoniae promote complete degradation of host-derived carbohydrate antigens

An important aspect of the interaction between the opportunistic bacterial pathogen Streptococcus pneumoniae and its human host is its ability to harvest host glycans. The pneumococcus can degrade a variety of complex glycans, including N- and O-linked glycans, glycosaminoglycans, and carbohydrate antigens, an ability that is tightly linked to the virulence of S. pneumoniae Although S. pneumoniae is known to use a sophisticated enzyme machinery to attack the human glycome, how it copes with fucosylated glycans, which are primarily histo-blood group antigens, is largely unknown. Here, we identified two pneumococcal enzymes, SpGH29C and SpGH95C, that target α-(1→3/4) and α-(1→2) fucosidic linkages, respectively. X-ray crystallography studies combined with functional assays revealed that SpGH29C is specific for the LewisA and LewisX antigen motifs and that SpGH95C is specific for the H(O)-antigen motif. Together, these enzymes could defucosylate LewisY and LewisB antigens in a complementary fashion. In vitro reconstruction of glycan degradation cascades disclosed that the individual or combined activities of these enzymes expose the underlying glycan structure, promoting the complete deconstruction of a glycan that would otherwise be resistant to pneumococcal enzymes. These experiments expand our understanding of the extensive capacity of S. pneumoniae to process host glycans and the likely roles of α-fucosidases in this. Overall, given the importance of enzymes that initiate glycan breakdown in pneumococcal virulence, such as the neuraminidase NanA and the mannosidase SpGH92, we anticipate that the α-fucosidases identified here will be important factors in developing more refined models of the S. pneumoniae-host interaction.</p

Xenobiotic-metabolizing enzymes in Bacillus anthracis: molecular and functional analysis of a truncated arylamine N-acetyltransferase isozyme

International audienceBACKGROUND AND PURPOSE The arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes that play an important role in the detoxification and/or bioactivation of arylamine drugs and xenobiotics. In bacteria, NATs may contribute to the resistance against antibiotics such as isoniazid or sulfamides through their acetylation, which makes this enzyme family a possible drug target. Bacillus anthracis, a bacterial species of clinical significance, expresses three NAT isozymes with distinct structural and enzymatic properties, including an inactive isozyme ((BACAN) NAT3). (BACAN) NAT3 features both a non-canonical Glu residue in its catalytic triad and a truncated C-terminus domain. However, the role these unusual characteristics play in the lack of activity of the (BACAN) NAT3 isozyme remains unclear. EXPERIMENTAL APPROACH Protein engineering, recombinant expression, enzymatic analyses with aromatic amine substrates and phylogenetic analysis approaches were conducted. KEY RESULTS The deletion of guanine 580 (G580) in the nat3 gene was shown to be responsible for the expression of a truncated (BACAN) NAT3 isozyme. Artificial re-introduction of G580 in the nat3 gene led to a functional enzyme able to acetylate several arylamine drugs displaying structural characteristics comparable with its functional Bacillus cereus homologue ((BACCR) NAT3). Phylogenetic analysis of the nat3 gene in the B. cereus group further indicated that nat3 may constitute a pseudogene of the B. anthracis species. CONCLUSION AND IMPLICATIONS The existence of NATs with distinct properties and evolution in Bacillus species may account for their adaptation to their diverse chemical environments. A better understanding of these isozymes is of importance for their possible use as drug targets

Crystallization and preliminary X-ray characterization of arylamine N-acetyltransferase C (BanatC) from Bacillus anthracis

Bacillus anthracis arylamine N-acetyltransferase C (BanatC) is an enzyme that metabolizes the drug sulfamethoxazole. Crystals of the purified enzyme that diffract at 1.95 Å are reported

Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3]

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å, β = 103.8°