Sequence determinants of nucleotide binding in Sucrose Synthase: improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues

Margo Diricks, Alexander Gutmann, Simon Debacker, Griet Dewitte, Bernd Nidetzky, Tom Desmet

Research output: Contribution to journalArticleResearchpeer-review

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).
Original languageEnglish
Pages (from-to)141-148
JournalProtein engineering, design and selection
Volume30
Issue number3
DOIs
Publication statusPublished - 2017

Fingerprint

Uridine Diphosphate
Sugar (sucrose)
Nucleotides
Administrative data processing
Diphosphates
Nucleosides
Adenosine Diphosphate
Nucleotide Motifs
Enzymes
Glycosylation
Flavonoids
Fructose
Aspalathus
Acidithiobacillus
Cytidine Diphosphate
Glucose
Recycling
Glycosyltransferases
Bacteria
Glucosides

Keywords

  • biocatalysis
  • carbohydrates
  • nucleotides
  • Sucrose Synthase
  • UDP-glucose recycling

ASJC Scopus subject areas

  • Biotechnology

Fields of Expertise

  • Human- & Biotechnology

Treatment code (Nähere Zuordnung)

  • Basic - Fundamental (Grundlagenforschung)

Cite this

Sequence determinants of nucleotide binding in Sucrose Synthase: improving the affinity of a bacterial Sucrose Synthase for UDP by introducing plant residues. / Diricks, Margo; Gutmann, Alexander; Debacker, Simon; Dewitte, Griet; Nidetzky, Bernd; Desmet, Tom.

In: Protein engineering, design and selection, Vol. 30, No. 3, 2017, p. 141-148.

Research output: Contribution to journalArticleResearchpeer-review

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AB - 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).

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