Crystal Structure of Levansucrase from the Gram-Negative Bacterium Brenneria Provides Insights into Its Product Size Specificity

Wei Xu, Dawei Ni, Xiaodong Hou, Tjaard Pijning, Albert Guskov, Yijian Rao, Wanmeng Mu*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Microbial levansucrases (LSs, EC 2.4.1.10) have been widely studied for the synthesis of β-(2,6)-fructans (levan) from sucrose. LSs synthesize levan-type fructo-oligosaccharides, high-molecular-mass levan polymer or combinations of both. Here, we report crystal structures of LS from the G-bacterium Brenneria sp. EniD 312 (Brs-LS) in its apo form, as well as of two mutants (A154S, H327A) targeting positions known to affect LS reaction specificity. In addition, we report a structure of Brs-LS complexed with sucrose, the first crystal structure of a G-LS with a bound substrate. The overall structure of Brs-LS is similar to that of G- and G+-LSs, with the nucleophile (D68), transition stabilizer (D225), and a general acid/base (E309) in its active site. The H327A mutant lacks an essential interaction with glucosyl moieties of bound substrates in subsite +1, explaining the observed smaller products synthesized by this mutant. The A154S mutation affects the hydrogen-bond network around the transition stabilizing residue (D225) and the nucleophile (D68), and may affect the affinity of the enzyme for sucrose such that it becomes less effective in transfructosylation. Taken together, this study provides novel insights into the roles of structural elements and residues in the product specificity of LSs.

Original languageEnglish
Pages (from-to)5095-5105
Number of pages11
JournalJournal of Agricultural and Food Chemistry
Volume70
Issue number16
Early online date7-Apr-2022
DOIs
Publication statusPublished - 27-Apr-2022

Keywords

  • crystal structure
  • fructooligosaccharides
  • levan
  • levansucrase
  • product specificity

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