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.