Bacterial sucrose isomerases: properties and structural studies

International audienceDue to their significant role in food industry, sucrose isomerases are good candidates for rational protein engineering. Hence, specific modi. cations in order to modify substrate affinity and selectivity, product specificity but also to adapt their catalytic properties to particular industrial process conditions, is interesting. Our work on the structural studies of the sucrose isomerase, MutB, which presents the first structural data available on a trehalulose synthase and the first experimental data on complexed forms of sucrose isomerases represents a significant advance in the understanding of these enzymes. In this review we give an overview of what is known on biochemical properties and structural aspects of different sucrose isomerases in particular those reported from bacteria

Probing the Role of Divalent Metal Ions in a Bacterial Psychrophilic Metalloprotease: Binding Studies of an Enzyme in the Crystalline State by X-Ray Crystallography

The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted ∼4, 1.0, and 1.6 Å, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions

Structure / function relationships of sucrose isomerases with different product specificity

Proceedings of the Symposium on Amylases and Related Enzymes, 2009International audienceSucrose isomerases from Protaminobacter rubrum, SmuA, and from Pseudomonas mesoacidophila MX-45, MutB, have been crystallized, and their three-dimensional structures solved. Determination of these crystal structures in their native states as well as in complex with substrate and substrate analogues have con- tributed to the visualization of a part of the double displacement reaction mechanism of this class of enzymes, and to the understanding of the specificity of the products. Comparative structural studies between the three- dimensional structures of trehalulose synthase, MutB, and the isomaltulose synthase, SmuA, have been conducted as well

The overexpression of the SAPB of Bacillus pumilus CBS and mutated sapB-L31I/T33S/N99Y alkaline proteases in Bacillus subtilis DB430: New attractive properties for the mutant enzyme

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