Levansucrases (EC 2.4.1.10) are
bacterial extracellular enzymes that are classified to family 68 of glycoside
hydrolases (http://www.cazy.org). They split sucrose and synthesize levan, a b-(2,6)-linked polyfructan. Structure-function
relationships of levansucrases from Pseudomonas
bacteria are poorly studied though they are promising catalysts for
biotechnology and their specific role in plant pathogenesis is not yet
completely solved [1, 2].
Levansucrase Lsc3 encoded by lsc3 (lsc-3) gene of Pseudomonas
syringae pv. tomato DC3000 is a promising catalyst for biotechnology
due to its high catalytic activity, stability and the ability to produce
several biotechnologically applicable products such as levan,
fructooligosaccharides (FOS) and heterooligofructans [1, 2, 3]. To initiate the structure-function study of Pseudomonas-derived
levansucrases, we conducted mutational analysis of the protein. We confirmed experimentally that Asp62,
Asp219 and Glu303 act as catalytic triad residues of Lsc3. To disclose other
catalytically important positions of the protein, conservation analysis of
levansucrases was performed. Residues from highly conserved regions adjacent to catalytic triad were
mutated site-specifically to reveal their potential functions. In total, sixteen single amino acid replacement mutants of twelve
different positions were constructed. The mutant proteins were purified and biochemically characterized by
determining affinity to substrates, catalytic efficiencies, FOS and levan
synthesis. In addition to catalytic triad, we revealed several positions of Lsc3
that are highly relevant for the catalysis and synthesis of reaction products.
Acknowledgements
This study was supported by grant GLOMR9072 from Estonian Science Foundation and EU project Functional Food Ingredients 3.2.071.12-0041 (SLOMR12215T) managed by Archimedes Foundation.
References
[1]
Visnapuu T. et
al, 2011, J Biotechnol 155, 338-49
[2]
Alamäe T. et al, 2012, Carbohydr Chem, Vol 38. RSC, Cambridge, UK, 176-91
[3]
Visnapuu, T. et al, 2009, Rapid
Commun Mass Spectrom 23, 1337-46
