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Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase.

ABSTRACT: Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.

SUBMITTER: Raga-Carbajal E

PROVIDER: S-EPMC6177408 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase.

Raga-Carbajal Enrique E López-Munguía Agustín A Alvarez Laura L Olvera Clarita C

Scientific reports 20181009 1

Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. T ...[more]

PMID: 30301900

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Project description: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.

Dataset Information

Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase.

Publications

Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase.

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