title: Sequence determinants of nucleotide binding in Sucrose Synthase: improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues.
abstract: Sucrose Synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate (NDP) into NDP-glucose and fructose. Biochemical characterization of several plant and bacterial SuSys has revealed that the eukaryotic enzymes preferentially use UDP whereas prokaryotic SuSys prefer ADP as acceptor. In this study, SuSy from the bacterium Acidithiobacillus caldus, which has a higher affinity for ADP as reflected by the 25-fold lower Km value compared to UDP, was used as a test case to scrutinize the effect of introducing plant residues at positions in a putative nucleotide binding motif surrounding the nucleobase ring of NDP. All eight single to sextuple mutants had similar activities as the wild-type enzyme but significantly reduced Km values for UDP (up to 60 times). In addition, we recognized that substrate inhibition by UDP is introduced by a methionine at position 637. The affinity for ADP also increased for all but one variant, although the improvement was much smaller compared to UDP. Further characterization of a double mutant also revealed more than 2-fold reduction in Km values for CDP and GDP. This demonstrates the general impact of the motif on nucleotide binding. Furthermore, this research also led to the establishment of a bacterial SuSy variant that is suitable for the recycling of UDP during glycosylation reactions. The latter was successfully demonstrated by combining this variant with a glycosyltransferase in a one-pot reaction for the production of the C-glucoside nothofagin, a health-promoting flavonoid naturally found in rooibos (tea).
authors: Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
authors: Margo
authors: M
authors: Diricks
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.
authors: Alexander
authors: A
authors: Gutmann
authors: Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
authors: Simon
authors: S
authors: Debacker
authors: Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
authors: Griet
authors: G
authors: Dewitte
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, 8010 Graz, Austria.|Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors: Bernd
authors: B
authors: Nidetzky
authors: Department of Biochemical and Microbial Technology, Centre for Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
authors: Tom
authors: T
authors: Desmet
mesh_terms: D042763:Acidithiobacillus / Q000201:enzymology / Q000235:genetics; D000244:Adenosine Diphosphate / Q000737:chemistry; D036903:Aspalathus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics; D014530:Uridine Diphosphate / Q000737:chemistry*
chemical_list: D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D005964:Glucosyltransferases; C022699:sucrose synthase
keywords: Sucrose Synthase; UDP-glucose recycling; biocatalysis; carbohydrates; nucleotides