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

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.

The molecular basis of the nonprocessive elongation mechanism in levansucrases

“…R246 has, in fact, a relevant role in maintaining LS activity ( 22 ), which is most likely due to its participation in coordinating donor substrates at subsite −1. On the other hand, many reports have already highlighted the participation of N242 in the polymerization reaction of gram-positive LSs, and its mutation to alanine, histidine, tyrosine, or tryptophan invariably limited the products to tetrasaccharides ( 12 , 17 , 19 , 33 ). Considering that the SacB mutant N242A also showed an increased K m, its deleterious effect may be related to the impaired coordination of sucrose as an acceptor molecule, thereby greatly affecting the formation of both HMW and LMW levans at the very beginning of the elongation process.…”

“…Furthermore, residues N242 and Y237 were identified as part of the +2 subsite, as they are responsible for only a few water-mediated interactions with the galactosyl unit from raffinose ( 15 ). Site-directed mutagenesis studies on LSs have revealed that modifications of residues N242, K363, and Y237 (numbering of B. subtilis SacB), which are located on the surface of the catalytic cavity, affect the enzyme catalytic efficiency, transfructosylation, and hydrolysis ratio by interrupting the polymerization process, and thus, these residues control the chain lengths of levans ( 12 , 16 , 17 , 18 , 19 ). However, the interactions mediated by these residues have not yet been identified, and the existence of additional external acceptor-binding subsites acting as structural determinants in the elongation of levans has not yet been clarified.…”

Computational design of SARS-CoV-2 peptide binders with better predicted binding affinities than human ACE2 receptor

“…In this study, a hydrogen bond was considered to occur if the following criteria were met: (1) a proton donor–acceptor distance ≤ 3.5 Å and (2) a donor-H-acceptor bond angle ≥ 120° 42 , 54 , 55 , 64 . Hydrogen bond occupations were defined into four levels: (1) strong hydrogen bonds (hydrogen bond occupations > 75%), (2) medium hydrogen bonds (75% ≥ hydrogen bond occupations > 50%), (3) weak hydrogen bond interactions (50% ≥ hydrogen bond occupations > 25%) and (4) very weak hydrogen bond interactions (25% ≥ hydrogen bond occupations > 5%) 42 , 55 , 56 . To compute peptide helicities, Define Secondary Structure of Protein (DSSP) was employed.…”

Unravelling Regioselectivity of Leuconostoc citreum ABK-1 Alternansucrase by Acceptor Site Engineering

“…LEaP module was employed to construct the structure of alternansucrase with glc-D635 intermediate. It was minimized with the five-step procedure [ 47 , 48 , 49 , 50 , 51 , 52 , 53 ]. Then, the system was heated, equilibrated and subsequently simulated for the production run at the experimental temperature of 310 K for 70 ns.…”