In Silico Study on Predicting Effects of H243L Mutation in Bacillus subtilis Levansucrase towards Sucrose Binding Affinity

Abstract:

Levan is a branched polysaccharide made from fructose monomers linked by β (2-6) and β (2-1) glycosidic bonds. Levan biosynthesis is catalyzed with levansucrase (EC 2.4.1.10) using sucrose as its substrate. In the present study, we are interested in investigating the elongation capability of the polymerase domain of the enzyme. The other study on Bacillus subtilis levansucrase found that the mutation of His243 into Leu has successfully prolonged the elongation of the polymer. The study, however, has not clearly accounted for the effect of the mutation. The current study aimed to employ in silico method to reveal the effect of the mutation. The initial model of substrate binding on the wild-type and H243L mutant levansucrase was prepared by molecular docking. The stability of the substrate-binding was evaluated by molecular dynamics simulation, while the binding affinity was calculated by the MM-PBSA method. The result showed that the H243L mutant was more stable in binding the substrate than the wild type. This was supported by the calculated binding affinity for H243L mutant and wild-type, which were −2.9 and +4,8 kcal/mol, respectively. Further molecular analysis suggested that mutation of His243 into Leu made the nearby Glu340 and Arg246 form additional hydrogen bonds with the substrate.

• Periodical: Materials Science Forum (Volume 1061)
• Pages: 119-127
• DOI: https://doi.org/10.4028/p-43hm23
• Online since: May 2022
• Authors: Jason Andrew Sudijanto, Rukman Hertadi*
• Keywords: Bacillus subtilis, Binding Affinity, Levansucrase, MM-PBSA, Molecular Docking, Molecular Dynamics (MD)

References

[1] R. Srikanth, C.H.S.S.S. Reddy, G. Siddartha, M.J. Ramaiah, K.B. Uppuluri, Review on production, characterization and applications of microbial levan, Carbohydr. Polym. 120 (2015) 102–114.

DOI: 10.1016/j.carbpol.2014.12.003

[2] R. Hertadi, M.M.S. Amari, E. Ratnaningsih, Enhancement of antioxidant activity of levan through the formation of nanoparticle systems with metal ions, Heliyon 6 (2020) e04111.

DOI: 10.1016/j.heliyon.2020.e04111

[3] S.J. Kim, P.K. Bae, B.H. Chung, Self-assembled levan nanoparticles for targeted breast cancer imaging, Chem. Commun. 51 (2015) 107–110.

DOI: 10.1039/c4cc07679f

[4] Á. González-Garcinuño, A. Tabernero, G. Marcelo, V. Sebastián, M. Arruebo, J. Santamaría, E.M. del Valle, Differences in levan nanoparticles depending on their synthesis route: Microbial vs cell-free systems, Int. J. Biol. Macromol. 137 (2019) 62–68.

DOI: 10.1016/j.ijbiomac.2019.06.128

[5] C.S. Hundschell, F. Jakob, A.M. Wagemans, Molecular weight dependent structure of the exopolysaccharide levan, Int. J. Biol. Macromol. 161 (2020) 398–405.

DOI: 10.1016/j.ijbiomac.2020.06.019

[6] J. Combie, Properties of levan and potential medical uses, ACS Symp. Ser. 934 (2006) 263–269.

[7] H. Yanase, M. Maeda, E. Hagiwara, H. Yagi, K. Taniguchi, K. Okamoto, Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase, J. Biochem. 132 (2002) 565–572.

DOI: 10.1093/oxfordjournals.jbchem.a003258

[8] M.E. Ortiz-Soto, C. Possiel, J. Görl, A. Vogel, R. Schmiedel, J. Seibel, Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation, Glycobiology 27 (2017) 755–765.

DOI: 10.1093/glycob/cwx050

[9] P. Phengnoi, T. Charoenwongpaiboon, K. Wangpaiboon, M. Klaewkla, S. Nakapong, W. Visessanguan, K. Ito, R. Pichyangkura, K. Kuttiyawong, Levansucrase from Bacillus Amyloliquefaciens KK9 and its Y237S variant producing the high bioactive levan-type fructooligosaccharides, Biomolecules 10 (2020) 692.

DOI: 10.3390/biom10050692

[10] C. He, Y. Yang, R. Zhao, J. Qu, L. Jin, L. Lu, L. Xu, M. Xiao, Rational designed mutagenesis of levansucrase from Bacillus licheniformis 8-37-0-1 for product specificity study, Appl. Microbiol. Biotechnol. 102 (2018) 3217–3228.

DOI: 10.1007/s00253-018-8854-3

[11] T. Sitthiyotha, R. Pichyangkura, S. Chunsrivirot, 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, PLoS One 13 (2018) e0204915.

DOI: 10.1371/journal.pone.0204915

[12] M.E. Ortiz-Soto, J.R. Porras-Domínguez, J. Seibel, A.L.M. López-Munguía, A close look at the structural features and reaction conditions that modulate the synthesis of low and high molecular weight fructans by levansucrases, Carbohydr. Polym. 219 (2019) 130–142.

DOI: 10.1016/j.carbpol.2019.05.014

[13] M.E. Ortiz-Soto, M. Rivera, E. Rudiño-Piñera, C. Olvera, A. López-Munguía, Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties, Protein Eng., Des. Sel. 21 (2008) 589–595.

DOI: 10.1093/protein/gzn036

[14] L.K. Ozimek, S. Kralj, M.J.E.C. van der Maarel, L. Dijkhuizen, The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions, Microbiology 154 (2006) 1187–1196.

DOI: 10.1099/mic.0.28484-0

[15] G. Meng, K. Fütterer, Structural framework of fructosyl transfer in Bacillus subtilis levansucrase, Nat. Struct. Mol. Biol. 10 (2003) 935–941.

DOI: 10.1038/nsb974

[16] W. Humphrey, A. Dalke, K. Schulten, VMD: Visual molecular dynamics, J. Mol. Graphics 14 (1996) 33–38.

DOI: 10.1016/0263-7855(96)00018-5

[17] R. Anandakrishnan, B. Aguilar, A.V. Onufriev, H++ 3.0: Automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations, Nucleic Acids Res. 40 (2012) W537–W541.

DOI: 10.1093/nar/gks375

[18] S. Pronk, S. Páll, R. Schulz, P. Larsson, P. Bjelkmar, R. Apostolov, M.R. Shirts, J.C. Smith, P.M. Kasson, D. Van Der Spoel, B. Hess, E. Lindahl, GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit, Bioinformatics 29 (2013) 845–854.

DOI: 10.1093/bioinformatics/btt055

[19] O. Trott, A.J. Olson, AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, J. Comput. Chem. 31 (2009) 455–461.

DOI: 10.1002/jcc.21334

[20] R.A. Laskowski, M.B. Swindells, LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery, J. Chem. Inf. Model. 51 (2011) 2778–2786.

DOI: 10.1021/ci200227u

[21] K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, O. Guvench, P. Lopes, I. Vorobyov, A.D. Mackerell, CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields, J. Comput. Chem. 31 (2010) 671–690.

DOI: 10.1002/jcc.21367

[22] M.S.V. Tresanco, M.E. Valdes-Tresanco, P.A. Valiente, E.M. Frías, gmx_MMPBSA (Version v1.4.2), https://zenodo.org/record/4814044#.Ycqej1kxVD8 (2021).

[23] B.R. Miller, T.D. McGee, J.M. Swails, N. Homeyer, H. Gohlke, A.E. Roitberg, MMPBSA.py: An efficient program for end-state free energy calculations, J. Chem. Theory Comput. 8 (2012) 3314–3321.

DOI: 10.1021/ct300418h

[24] J. Seibel, R. Moraru, S. Götze, K. Buchholz, S. Na'amnieh, A. Pawlowski, H.J. Hecht, Synthesis of sucrose analogues and the mechanism of action of Bacillus subtilis fructosyltransferase (levansucrase), Carbohydr. Res. 341 (2006) 2335–2349.

DOI: 10.1016/j.carres.2006.07.001

[25] M. Okuyama, R. Serizawa, M. Tanuma, A. Kikuchi, J. Sadahiro, T. Tagami, W. Lang, A. Kimura, Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and β-fructofuranosidase, J. Biol. Chem. 296 (2021) 100398.

DOI: 10.1016/j.jbc.2021.100398

[26] A. Homann, R. Biedendieck, S. Götze, D. Jahn, J. Seibel, Insights into polymer versus oligosaccharide synthesis: Mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium, Biochem. J. 407 (2007) 189–198.

DOI: 10.1042/bj20070600