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.
Highlights
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)
These findings suggest that transfructosylation should be able to occur in these systems, i.e., the wild type, the N251A and N251Y mutants should be able to extend GF2 by one fructosyl residue to create GF3
Molecular dynamics (MD) was performed on the GF2-LSwt, GF2-LSN251A, GF2-LSN251Y, GF3-LSwt, GF3LSN251A 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
Summary
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). Levan and levan oligosaccharides can be used as a prebiotic ingredient [5], encapsulating agent, emulsifier, thickener [3] and cholesterol lowering agent [6]. They can be used as a component in cosmetics to alleviate skin irritation and moisturize skin [7]. Glu351 acts as a general base that removes a proton from O6 of the non-reducing end of the acceptor This O6 attacks the fructosyl C2 of the covalent fructosyl-enzyme intermediate, creating the β-(2, 6) linkage to extend the levan chain. The bond between the fructosyl residue and Asp is broken, and the product is released [13]
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