A novel levansucrase was identified in the supernatant of a cell culture of Bacillus megaterium DSM319. In order to test for the contribution of specific 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 characterized 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 specificity and catalysis. Highly conserved regions such as the previously described RDP and DXXER motifs were identified as being important. Two crucial structural differences localized at amino acid residues Arg370 and Asn252 were of high relevance in polymer compared with oligosaccharide synthesis.
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October 2007
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Research Article|
September 25 2007
Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium
Arne Homann;
Arne Homann
1
*Department of Technical Chemistry, Technical University Braunschweig, Section of Carbohydrate Technology, Hans-Sommer-Str. 10, D-38106 Braunschweig, Germany
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Rebekka Biedendieck;
Rebekka Biedendieck
1
†Department of Microbiology, Technical University Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany
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Sven Götze;
Sven Götze
*Department of Technical Chemistry, Technical University Braunschweig, Section of Carbohydrate Technology, Hans-Sommer-Str. 10, D-38106 Braunschweig, Germany
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Dieter Jahn;
Dieter Jahn
†Department of Microbiology, Technical University Braunschweig, Spielmannstr. 7, D-38106 Braunschweig, Germany
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Jürgen Seibel
Jürgen Seibel
2
*Department of Technical Chemistry, Technical University Braunschweig, Section of Carbohydrate Technology, Hans-Sommer-Str. 10, D-38106 Braunschweig, Germany
2To whom correspondence should be addressed (email j.seibel@tu-bs.de).
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Publisher: Portland Press Ltd
Received:
May 04 2007
Revision Received:
June 14 2007
Accepted:
July 04 2007
Accepted Manuscript online:
July 04 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 407 (2): 189–198.
Article history
Received:
May 04 2007
Revision Received:
June 14 2007
Accepted:
July 04 2007
Accepted Manuscript online:
July 04 2007
Citation
Arne Homann, Rebekka Biedendieck, Sven Götze, Dieter Jahn, Jürgen Seibel; Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochem J 15 October 2007; 407 (2): 189–198. doi: https://doi.org/10.1042/BJ20070600
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