Amino acid substitutions of His296 alter the catalytic properties of Zymomonas mobilis 10232 levansucrase
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His296 of Zymomonas mobilis levansucrase (EC 220.127.116.11) is crucial for the catalysis of the transfructosylation reaction. The three-dimensional structures of levansucrases revealed the His296 is involved in the substrate recognition and binding. In this study, nine mutants were created by site-directed mutagenesis, in which His296 was substituted with amino acids of different polarity, charge and length. The substitutions of His296 with Arg or Trp retained partial hydrolysis and transfructosylation activities. The positively charged Lys substitution resulted in a 2.5-fold increase of sucrose hydrolysis. Substitutions with short (Cys or Ser), negatively charged (Glu) or polar (Tyr) amino acids virtually abolished both the activities. Analysis of transfructosylation products indicated that the mutants synthesized different oligosaccharides, suggesting that amino acid substitutions of His296 strongly affected both the enzyme activity and transfructosylation products.
- Ananthalakshmy VK, Gunasekaran P (1999) Overproduction of levan in Zymomonas mobilis by using cloned sacB gene. Enzyme Microb Technol 25: 109-115.
- Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
- Chambert R, Petit-Glatron MF (1991) Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis. Biochem J 279: 35-41.
- Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723.
- Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280: 309-316.
- Kazap I, Leibovici J, Wolman M (1980) Blood levels of high-molecular levan in mice as a function of the route and duration of administration. Arzneimittelforschung 30: 459-462.
- Martinez-Fleites C, Ortiz-Lombardia M, Pons T, Tarbouriech N, Taylor EJ, Arrieta JG, Hernandez L, Davies GJ (2005) Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus. Biochem J 390: 19-27.
- Meng G, Futterer K (2003) Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol 10: 935-941.
- Sarkar G, Sommer SS (1990) The 'megaprimer' method of site-directed mutagenesis. Biotechniques 8: 404-407.
- Senthilkumar V, Busby SJ, Gunasekaran P, Senthikumar V, Bushby SJ (2003) Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity. Biotechnol Lett 25: 1653-1656.
- Somogyi M (1952) Notes on sugar determination. J Biol Chem 195: 19-23.
- Swings J, De Ley J (1977) The biology of Zymomonas. Bacteriol Rev 41: 1-46.
- Yamamoto Y, Takahashi Y, Kawano M, Iizuka M, Matsumoto T, Saeki S, Yamaguchi H (1999) In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats. J Nutr Biochem 10: 13-18.
- Yanase H, Iwata M, Nakahigashi R, Kita K, Kato N, Tonomura K (1992) Purification, crystallization and properties of the extracellular levansucrase from Zymomonas mobilis. Biosci Biotechnol Biochem 56: 1335-1337.
- Yanase H, Maeda M, Hagiwara E, Yagi H, Taniguchi K, Okamoto K (2002) Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase. J Biochem (Tokyo) 132: 565-572.
- Yun JW (1996) Fructooligosaccharides - occurrence, preparation and application. Enzyme Microb Technol 19: 107-117.
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