Structural Modelling and Structure-Function Analysis of Zymomonas mobilis Levansucrase

Levansucrases are bacterial enzymes which produce fructan polymers from sucrose via hydrolysis and transfructosylation activities. These polymers; levan and fructooligosaccharides are valuable for food and pharmaceutical industries. Levansucrases from Gram-positive bacteria such as Bacillus subtilis tend to produce levan, while those from Gram-negative bacteria preferentially produce fructooligosaccharides. Zymomonas mobilis is an efficient levansucrase producer and its extracellular levansucrase can produce both fructooligosaccharides and levan depending on the reaction parameters. In this study, the structure of Z. mobilis levansucrase was modeled in order to help to understand the structure-function relationship of the enzyme. Furthermore, amino acids previously reported to be important for levansucrase activity were mapped on the model. The structural model presents a five-bladed propeller with a deep, negatively charged central pocket, similar to other bacterial levansucrases. Mapping showed that amino acids which previously reported to affect fructan length are located on the periphery of the structure covering the active site central pocket. Thus it is showed that, for the first time, that hydrolysis and transfructosylation reactions are catalyzed on different parts of Z. mobilis levansucrase structure. The structural location of the critical amino acids will pave the way to identify other residues which control fructan length by site directed mutagenesis without altering the overall fold of the enzyme.

Anahtar Kelimeler:

Levansucrase, Zymomonas mobilis; Protein structure modelling; Structural bioinformatics

PDF

___

[1] Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 280(2), 309-316.
[2] Sabater-Molina, M., Larquee, E., Torrella, F., Zamora, S. 2009. Dietary fructooligosaccharides and potential benefits on health. Journal of Physiology and Biochemistry, 65(3), 315-328.
[3] İnanç, N., Şahin, H., Çiçek, B. 2005. Probiyotik ve Prebiyotiklerin Sağlık Üzerine Etkileri. Erciyes Tıp Dergisi, 27(3), 122-127.
[4] Yun, W. Y. 1996. Fructooligosaccharides-Occurrence, preparation, and application. Enzyme and Microbial Technology, 19(2), 107-117.
[5] Kim, K. H., Chung, C . B., Kim, Y. H., Kim, K. S., Han, C. S., Kim, C. H. 2005. Cosmeceutical Properties of Levan Produced by Zymomonas mobilis. Journal of Cosmetic Science, 56(6), 395-406.
[6] Öner, E. T., Hernandez, L., Combie, J. 2016. Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnology Advances, 34(5), 827–844.
[7] Byun, B. Y., Lee S. J., Mah, J. H. 2014. Antipathogenic activity and preservative effect of levan (β-2,6-fructan), a multifunctional polysaccharide. International Journal of Food Science & Technology, 49(1), 238–245.
[8] Chambert, R., Gonzy-Treboul, G. 1976. Levansucrase of Bacillus subtilis: Kinetic and Thermodynamic Aspects of Transfructosylation Process. European Journal of Biochemistry, 62(1), 55-64
[9] Yanase, H., Iwata, M., Nakahigashi, R., Kita, K., Kato, N., Tonomura, K. 1992. Purification, crystallization and properties of the extracellular levansucrase from Zymomonas mobilis. Bioscience Biotechnology and Biochemistry, 56 (8), 1335– 1337
[10] Meng, G., Fütterer, K. 2003. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nature Structural Biology, 10(11), 935–941
[11] Martinez-Fleites, C., Ortíz-Lombardía, M., Pons, T., Tarbouriech, N., Taylor, E. J., Arrieta, J. G., Davies, G. J. 2005. Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus. The Biochemical Journal, 390(1), 19–27.
[12] Wuerges, J., Caputi, L., Cianci, M., Boivin, S., Meijers, R., Benini, S. 2015. The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site. Journal of Structural Biology, 191(3), 290-298.
[13] Yanase, H., Maeda, M., Hagiwara, E., Yagi, H., Taniguchi, K., Okamoto, K. 2002. Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase. Journal of Biochemistry, 132(4), 565–572.
[14] Chambert, R., Treboul, G., Dedonder, R. 1974. Kinetic studies of levansucrase of Bacillus subtilis. European Journal of Biochemistry, 41(2), 285–300.
[15] Song, D. D., Jacques, N. A. 1999. Purification and enzymic properties of the fructosyltransferase of Streptococcus salivarius ATCC 25975. Biochemical Journal, 341(2), 285–291.
[16] Homann, A., Biedendieck, R., Götze, S., Jahn, D., Seibel, J. 2007. Insights into polymer versus oligosaccharide synthesis: mutagenesis and mechanistic studies of a novel levansucrase from Bacillus megaterium. Biochemical Journal, 407(2), 189–198.
[17] Hernandez, L., Arrieta, J., Menendez, C., Vazquez, R., Coego, A., Suarez, V., Selman, G., Petit-Glatron, M. F., Chambert, R. 1995. Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugarcane. Biochemical Journal, 309(1), 113–118.
[18] Caputi, L., Nepogodiev, S. A., Malnoy, M., Rejzek, M., Field, R. A., Benini, S. 2013. Biomolecular characterization of the levansucrase of Erwinia amylovora, a promising biocatalyst for the synthesis of fructooligosaccharides. Journal of Agricultural and Food Chemistry, 61(50), 12265–12273.
[19] Visnapuu, T., Mardo, K., Alamae, T. 2015. Levansucrases of a Pseudomonas syringaepathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides. New Biotechnology, 32(6), 597–605.
[20] Lammens, W., Le Roy, K., Schroeven, L., Van Laere, A., Rabijns, A. and Van den Ende, W. 2009. Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications. Journal of Experimental Botany, 60(3), 727–740
[21] Tanaka T., Oi, S., Yamamoto T. 1980. The molecular structure of low and high molecular weight levans synthesized by levansucrase, Journal of Biochemistry, 87(1), 297–303.
[22] Vigants, A., Upite, D., Scherbaka, R., Lukjanenko, J., Ionina, R. 2013. An influence of ethanol and temperature on products formation by different preparations of Zymomonas mobilis extracellular levansucrase. Folia Microbiologica, 58(1), 75–80.
[23] Santos-Moriano, P., Fernandez-Arrojo, L., Poveda, A., Jimenez-Barbero, J., Ballesteros, A. O., Plou, F. J. 2015. Levan versus fructooligosaccharide synthesis using the levansucrase from Zymomonas mobilis: effect of reaction conditions. Journal of Molecular Catalysis B: Enzymatic, 119, 18–25.
[24] Senthikumar, V., Bushby S. J. W., Gunasekaran, P. 2003. Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity. Biotechnology Letters, 25(19), 1653–1656
[25] Li, S. Y., Chen, M., Li, G., Yan, Y. L. , Yu, H. Y., Zhan, Y. H., Peng, Z. X., Wang, J., Lin, M. 2008. Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase. Acta Biochimica Polonica, 55(1), 201-206.
[26] Armougom F., Moretti, S., Keduas, V., Notredame, C. 2006. APDB: a web server to evaluate the accuracy of sequence alignments using structural information. Bioinformatics, 22(19), 35-39.
[27] Robert, X., Gouet, P. 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(1), 320-324.
[28] Kallberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., Xu, J. 2012. Template-based protein structure modeling using the RaptorX web server. Nature Protocols, 7(8) 1511–1522.
[29] Roy, A., Kucukural, A., Zhang, Y. 2010. I-TASSER: a unified platform for automated protein structure and function prediction, Nature Protocols, 5(4), 725-738.
[30] Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., Zhang. Y. 2015. The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12(1), 7-8.
[31] Yang, J., Roy, A., Zhang, Y. 2013. Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics (Oxford Journals), 29(20), 2588–2595.
[32] Chambert, R., Petit-Glatron, M. F. 1991. Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis. Biochemical Journal, 279(1), 35-41.