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.
Keywords: 2) fucosidase; 4-nitrophenyl-α-l-fucopyranoside; Amp, ampicillin; BHI, brain heart infusion medium; CBB, Coomassie brilliant blue G 250; DFJ, deoxyfuconojirimycin; Erm, erythromycin; FDH, fucose dehydrogenase; HPAEC, high-performance anion-exchange chromatography with pulsed amperometric detection; LC-ESI-MS, liquid chromatography-electrospray ionisation-mass spectrometry; NAM, N-acetylmuramic acid; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; T. forsythia, Tannerella forsythia ATCC 43037; TfFuc1, T. forsythia ATCC 43037 fucosidase-1 encoded by the bfo_2737 gene, equally Tffuc1; WT, wild-type bacterium.; enzyme activity; enzyme specificity; oral pathogen; pNP-fucose; periodontitis; rTfFuc-1, recombinant TfFuc1 enzyme; tannerella forsythia; α(1.