Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium

Arne Homann , Rebekka Biedendieck , Sven Götze , Dieter Jahn , Jürgen Seibel , Jürgen Seibel (1)

1 Department for Carbohydrate Technology

A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of these amino acid residues to levansucrase catalysis the wild-type enzyme along with 16 variants based on sequence alignments and structural information were heterologously produced in Escherichia coli. The purified enzymes were characterised kinetically and the product spectrum of each variant was determined. Comparison of the X-ray structures of the levansucrases from Gram positive Bacillus subtilis and Gram negative Gluconacetobacter diazotrophicus in conjunction with the corresponding product spectra identified crucial amino acid residues responsible for product specifity and catalysis. Highly conserved regions like the previously described RDP and DXXER motifs were identified to be important. Two crucial structural differences localised at the amino acid residues Arg 370} and Asn 252} were of high relevance in the differential polymer versus oligosaccharide synthesis.

Mots clés: Life Sciences

Arne Homann, Rebekka Biedendieck, Sven Götze, Dieter Jahn, Jürgen Seibel, et al.. Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochemical Journal, 2007, 407 (2), pp.189-198. ⟨10.1042/BJ20070600⟩. ⟨hal-00478795⟩