Research Article: 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

Date Published: October 2, 2018

Publisher: Public Library of Science

Author(s): Thassanai Sitthiyotha, Rath Pichyangkura, Surasak Chunsrivirot, Alexandre G. de Brevern.

http://doi.org/10.1371/journal.pone.0204915

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.

Partial Text

Levan and levan oligosaccharides (GFn) are natural fructans that contain one terminal glucopyranosyl residue and D-fructofuranosyl repeating unit linked by β-(2, 6) linkage in a main chain with some possible branching points linked by β-(2, 1) linkages [1] (Fig 1A). Properties of levan and levan oligosaccharides depend on their lengths and branching degrees [2], and they have various beneficial properties such as high-water solubility [3] and low intrinsic viscosity [4] for food, cosmetics and pharmaceutical industries. In the food industry, levan and levan oligosaccharides can be used as a prebiotic ingredient [5], encapsulating agent, emulsifier, thickener [3] and cholesterol lowering agent [6]. They can also be used as a component in cosmetics to alleviate skin irritation and moisturize skin [7]. For pharmaceutical industry, they could potentially be used as anti-tumor, anti-inflammatory and anti-viral agents [8].

In this work, MD was performed on the GF2-LSwt, GF2-LSN251A, GF2-LSN251Y, GF3-LSwt, GF3-LSN251A and GF3-LSN251Y complexes to gain insight into the effects of N251A and N251Y mutations on the binding of GF2/GF3 in the active site of Bacillus licheniformis RN-01 levansucrase. Our results of binding free energies and hydrogen bond occupations as well as the distances between atoms necessary for transfructosylation of GF3-LSwt, GF3-LSN251A and GF3-LSN251Y complexes support the hypothesis that these mutations reduced GF3 binding affinity in active site of levansucrase with fructosyl-Asp93 intermediate and caused GF3 to be in an unfavorable orientation for transfructosylation; therefore, transfructosylation could not occur in GF3-LSN251A and GF3-LSN251Y complexes. As a result, only the wild type should be able to extend GF3 by one fructosyl residue to produce GF4, supporting the experimental results that the wild type can produce GF4, but the N251A and N251Y mutants cannot effectively produce GF4. However, these mutations did not drastically change binding affinity or orientation of GF2 as shown by the binding free energy and hydrogen bond occupation results as well as the distances between atoms necessary for transfructosylation of GF2-LSwt, GF2-LSN251A and GF2-LSN251Y complexes. Therefore, the wild type, the N251A and N251Y mutants should be able to extend GF2 by one fructosyl residue to produce GF3, supporting the experimental results that the wild type, the N251A and N251Y mutants can produce GF3. Moreover, the free energy decomposition results also suggest the importance of Arg255 in GF2/GF3 binding in the active site of the wild type. Our results also show that Arg255 formed hydrogen bond networks with GF2/GF3, Asn251 and Glu349 in the wild-type complexes at the beginning of the 80 ns MD simulations, and the N251A and N251Y mutations disrupted these hydrogen bond networks. Although these hydrogen bond networks were disrupted in the GF2-LsN251A and GF2-LsN251Y complexes, GF2 could still bind in a favorable orientation for transfructosylation in the active sites of these mutants probably because there were other residues binding and forming hydrogen bonds with GF2, and these interactions helped prevent misorientation of GF2. However, GF3 could not bind in a favorable orientation for transfructosylation in the active sites of these mutants because there was significantly less number of residues binding and forming hydrogen bonds with GF3 in the mutant complexes than that in the wild-type complex. Our study provides important and novel insight into the binding of GF2/ GF3 in the active site of Bacillus licheniformis RN-01 levansucrase and into how N251A and N251Y mutations may disrupt production of long-chain levan.

 

Source:

http://doi.org/10.1371/journal.pone.0204915

 

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