Characterization of an α-l-fucosidase from the periodontal pathogen Tannerella forsythia.

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 µM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase.

Expression, purification and preliminary crystallographic analysis of Drosophila melanogaster lysosomal α-mannosidase.

The lysosomal α-mannosidases are class II mannosidases that belong to glycoside hydrolase family 38 and play an important role in the degradation of asparagine-linked carbohydrates of glycoproteins. Based on peptide similarity to human and bovine lysosomal mannosidase (LM), recombinant α-mannosidase from Drosophila melanogaster (dLM408) was cloned and heterologously expressed in Pichia pastoris. The recombinant form of dLM408 designed for structural analysis lacks the transmembrane domain and was crystallized using standard vapour-diffusion and counter-diffusion techniques. The crystals grew as flat plates and as tetragonal bipyramids, respectively. The plate-shaped crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 3.5 Å.

The never-ending story of peptide O-xylosyltransferase.

In a journey lasting 40 years from the first reports on its activity in the 1960s to its purification and the cloning of relevant complementary DNAs, peptide O-xylosyltransferase has finally arrived at the same point as many other enzymes. This enzyme, whose systematic name is UDP-alpha-D-xylose:proteoglycan core protein beta-D-xylosyltransferase (EC 2.4.2.26), catalyses the first step in the biosynthesis of chondroitin, dermatan and heparan sulphates in the endoplasmic reticulum and/or the cis-Golgi cisternae. Analyses of their primary structure show that peptide O-xylosyltransferases are members of glycosyltransferase family 14 and so are homologous to beta1,6- N-acetylglucosaminyltransferases involved in O-glycan and poly- N-acetyllactosamine branching. Furthermore, vertebrates appear to have two rather similar genes encoding xylosyltransferase I and xylosyltransferase II, but enzymatic activity was only detected for a recombinant form of the first isoform. On the other hand, Caenorhabditis and Drosophila have each only one peptide O-xylosyltransferase gene. In the worm sqv-6 mutant, a mutation of the xylosyltransferase gene is associated with defective vulval morphogenesis, indicative of the importance of the glycosaminoglycan chains of proteoglycans in animal development. There remain, however, open questions, for instance, on the enzyme's intracellular localisation and structure-function relationships.