Cloning and functional characterization of an α-1,3-fucosyltransferase from Bacteroides fragilis

• Published in: Biotechnology and bioprocess engineering Vol. 18; no. 5; pp. 843 - 849
• Main Authors: Lee, Joo-Ho, Pandey, Ramesh Prasad, Kim, DaeHee, Sohng, Jae Kyung
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.09.2013; Springer Berlin Heidelberg; 한국생물공학회
• Subjects: Azospirillum; Bacteroides fragilis; Biotechnology; Chemistry; Chemistry and Materials Science; gastrointestinal system; genes; Helicobacter pylori; Industrial and Production Engineering; molecular cloning; nucleotide sequences; pathogens; polymerase chain reaction; Research Paper; Rickettsia; Rickettsia bellii; spectroscopy; thin layer chromatography; 생물공학; Lewis X; acetyllactosamine; fucosyltransferases
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-013-0041-x

Bacteroides fragilis is a clinically important anaerobic pathogen present in the human gastrointestinal tract and is involved in a high number of anaerobic peritoneal infections. The complete genome sequence of B. fragilis NCTC 9343 revealed the presence of several putative fucosyltransferase gene homologues known as alpha-1,3-fucosyltransferases (α-1,3-FucTs). However, their expression and functional activities have not been studied. Here, we report the molecular cloning, functional expression, and characterization of the alpha-1,3-fucosyltransferase 3 (α-1,3-FucT3) enzyme from B. fragilis NCTC 9343. The polymerase chain reaction (PCR)-based approach was used to clone the 331 amino acid long (MW, ∼39 kDa) PCR product encoding fucosyltransferase enzyme. The enzyme had low identity of 30–40% with other known α-1,3-FucTs from Azospirillum sp, Rickettsia bellii, and different strains of Helicobacter pylori. An in vitro enzyme reaction analysis showed the ability of the enzyme to transfer the fucose moiety from guanosine-5′-diphosphate β-L-fucose to the N-acetyllactosamine to produce Lewis X. The reaction product, Lewis X was confirmed by thin layer chromatography, liquid chromatography-mass spectroscopy, and ¹H-nuclear magnetic resonance analyses.