Molecular dynamics provides insight into how N251A and N251Y mutations in the active site of Bacillus licheniformis RN-01 levansucrase disrupt production of long-chain levan

PLoS One. 2018 Oct 2;13(10):e0204915. doi: 10.1371/journal.pone.0204915. eCollection 2018.

Abstract

Produced by levansucrase, levan and levan oligosaccharides (GFn) have potential applications in food and pharmaceutical industries such as prebiotics, anti-tumor and anti-inflammatory agents. Previous study reported that Bacillus licheniformis RN-01 levansucrase could produce levan oligosaccharides and long-chain levan. However, its N251A and N251Y mutants could effectively produce short-chain oligosaccharides upto GF3, but they could not produce long-chain levan. We hypothesized that these mutations probably reduced GF3 binding affinity in levansucrase active site that contains fructosyl-Asp93 intermediate and caused GF3 to be in an unfavorable orientation for transfructosylation; therefore, levansucrase could not effectively extend GF3 by one fructosyl residue to produce GF4 and subsequently long-chain levan. However, these mutations probably did not significantly reduce binding affinity or drastically change orientation of GF2; therefore, levansucrase could still extend GF2 to produce GF3. Using this hypothesis, we employed molecular dynamics to investigate effects of these mutations on GF2/GF3 binding in levansucrase active site. Our results reasonably support this hypothesis as N251A and N251Y mutations did not significantly reduce GF2 binding affinity, as calculated by MM-GBSA technique and hydrogen bond occupations, or drastically change orientation of GF2 in levansucrase active site, as measured by distance between atoms necessary for transfructosylation. However, these mutations drastically decreased GF3 binding affinity and caused GF3 to be in an unfavorable orientation for transfructosylation. Furthermore, the free energy decomposition and hydrogen bond occupation results suggest the importance of Arg255 in GF2/GF3 binding in levansucrase active site. This study provides important and novel insight into the effects of N251A and N251Y mutations on GF2/GF3 binding in levansucrase active site and how they may disrupt production of long-chain levan. This knowledge could be beneficial in designing levansucrase to efficiently produce levan oligosaccharides with desired length.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus licheniformis / enzymology*
  • Bacillus licheniformis / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Binding Sites
  • Catalytic Domain
  • Fructans / metabolism
  • Hexosyltransferases / chemistry*
  • Hexosyltransferases / genetics
  • Hexosyltransferases / metabolism*
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Mutation*
  • Oligosaccharides / metabolism
  • Protein Binding

Substances

  • Bacterial Proteins
  • Fructans
  • Oligosaccharides
  • levan
  • Hexosyltransferases
  • levansucrase

Grants and funding

This research is supported by the Institute for the Promotion of Teaching Science and Technology (IPST) under the Research Fund for DPST Graduate with First Placement [Grant no. 07/2557 to SC] and Structural and Computational Biology Research Group, Special Task Force for Activating Research (STAR), Faculty of Science, Rachadaphiseksomphot Endowment Fund, Chulalongkorn University. TS was partially supported by the Scholarship from Graduate School, Chulalongkorn University to commemorate The Celebrations on the Auspicious Occasion of Her Royal Highness Princess Maha Chakri Sirindhorn's 5th Cycle (60th) Birthday. RP was partially supported by Rachadaphiseksomphot Endowment Fund Part of the "Strengthen CU's Researcher's Project." SC was also partially supported by the Ratchadaphiseksomphot Endowment Fund Part of the "Research Grant for New Scholar CU Researcher's Project," Chulalongkorn University [Grant no. RGN_2558_002_01_23] and the Thailand Research Fund [Grant no. TRG5880222 and IRG 5780008]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.