Two active forms of Zymomonas mobilis levansucrase. An ordered microfibril structure of the enzyme promotes levan polymerization.
Fructansucrases, members of glycoside hydrolase family 68, catalyze both sucrose hydrolysis and the polymerization of fructose to beta-d-fructofuranose polymers. The resulting fructan polymers are distinguished by the nature of the glycosidic bond: inulin (beta-(2-1)-fructofuranose) and levan (beta-(2-6)-fructofuranose). In this study we demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pH values above 7.0, the enzyme is mainly a dimer, whereas at pH values below 6.0, the protein forms well ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructosaccharides (degree of polymerization, 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of six to eight protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils with organized fibril networks and to synthesize levan at pH 6.0.
Goldman
Dan
D
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Lavid
Noa
N
Schwartz
Alon
A
Shoham
Gil
G
Danino
Dganit
D
Shoham
Yuval
Y
D002384:Catalysis; D003001:Cloning, Molecular; D019281:Dimerization; D005630:Fructans / Q000737:chemistry*; D006602:Hexosyltransferases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D020894:Microfibrils; D008956:Models, Chemical; D011108:Polymers / Q000737:chemistry; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry; D017355:Zymomonas / Q000201:enzymology*
D005630:Fructans; D011108:Polymers; D013395:Sucrose; C072599:levan; D006602:Hexosyltransferases; C021513:levansucrase