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

Produced by levansucrase, levan and levan oligosaccharides (GF(n)) 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 GF(3,) but they could not produce long-chain levan. We hypothesized that these mutations probably reduced GF(3) binding affinity in levansucrase active site that contains fructosyl-Asp93 intermediate and caused GF(3) to be in an unfavorable orientation for transfructosylation; therefore, levansucrase could not effectively extend GF(3) by one fructosyl residue to produce GF(4) and subsequently long-chain levan. However, these mutations probably did not significantly reduce binding affinity or drastically change orientation of GF(2); therefore, levansucrase could still extend GF(2) to produce GF(3). Using this hypothesis, we employed molecular dynamics to investigate effects of these mutations on GF(2)/GF(3) binding in levansucrase active site. Our results reasonably support this hypothesis as N251A and N251Y mutations did not significantly reduce GF(2) binding affinity, as calculated by MM-GBSA technique and hydrogen bond occupations, or drastically change orientation of GF(2) in levansucrase active site, as measured by distance between atoms necessary for transfructosylation. However, these mutations drastically decreased GF(3) binding affinity and caused GF(3) to be in an unfavorable orientation for transfructosylation. Furthermore, the free energy decomposition and hydrogen bond occupation results suggest the importance of Arg255 in GF(2)/GF(3) binding in levansucrase active site. This study provides important and novel insight into the effects of N251A and N251Y mutations on GF(2)/GF(3) 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.