Zymomonas mobilis Levansükrazının Yapısal Modellemesi ve Yapı‐Fonksiyon Analizi

Bakteriyal enzimler olan levansükrazlar, hidroliz ve transfruktosilasyon aktiviteleri ile sakkarozdan fruktan polimer oluşumunu katalizlerler. Bu polimerler; levan ve fruktooligosakkaritler, gıda ve ilaç endüstrileri için değerlidir. Bacillus subtilis gibi Gram‐pozitif bakterilerden elde edilen levansükrazlar levan üretme eğilimindeyken, Gram‐negatif bakteriler tercihen fruktooligosakkaritleri üretirler. Zymomonas mobilis etkili bir levansükraz üreticisi olup, hücre dışı levansükraz enzimi, tepkime parametrelerine bağlı olarak hem fruktooligosakkaritleri hem de levanı üretebilir. Bu çalışmada, enzimin yapı‐ fonksiyon ilişkisini anlamaya yardımcı olmak için Z. mobilis levansükrazın yapısı modellenmiş ve daha önce enzim aktivitesi için önemli olduğu bildirilen amino asitler model üzerinde haritalanmıştır. Elde edilen yapısal model, diğer bakteriyel levansükrazlara benzer şekilde derin, negatif yüklü bir merkezi cebe sahip beş bıçaklı pervane yapısına sahiptir. Amino asit haritalaması, daha önce fruktan uzunluğunu etkilediği bildirilen amino asitlerin, aktif bölge merkez cebini çevreleyecek şekilde yapının yüzeyinde bulunduğunu göstermiştir. Böylece, bu çalışma ile ilk kez, hidroliz ve transfruktosilasyon tepkimelerinin Z. mobilis levansükraz yapısının farklı kısımlarında katalizlendiği gösterilmiştir. Kritik amino asitlerin yapısal konumunun bilinmesi, fruktan uzunluğunu kontrol eden diğer amino asitlerin mutajenez yoluyla ve protein yapısına zarar vermeden belirlenmesine yardımcı olacaktır.

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.

PDF

___

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.
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.
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.
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.
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.
Robert, X., Gouet, P. 2014. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Research, 42(1), 320‐324.
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.
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.
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
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.
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.
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.
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
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.
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.
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.
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.
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.
Chambert, R., Treboul, G., Dedonder, R. 1974. Kinetic studies of levansucrase of Bacillus subtilis. European Journal of Biochemistry, 41(2), 285–300.
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.
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.
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.
Meng, G., Fütterer, K. 2003. Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nature Structural Biology, 10(11), 935–941
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
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
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.
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.
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.
Yun, W. Y. 1996. Fructooligosaccharides‐ Occurrence, preparation, and application. Enzyme and Microbial Technology, 19(2), 107‐117.
İnanç, N., Şahin, H., Çiçek, B. 2005. Probiyotik ve Prebiyotiklerin Sağlık Üzerine Etkileri. Erciyes Tıp Dergisi, 27(3), 122‐127.
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.
Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 280(2), 309‐316.