New Structure of Class II Fructose-1,6-Bisphosphatase from Francisella tularensis suggest a novel catalytic mechanism for the entire class

Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of enzymes from different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product and divalent metal cations in the enzyme catalytic centers led to a revision of the catalytic mechanism suggested previously for class II FBPases. Instead of a metal cofactor for the stabilization of the transition state of the leaving phosphate group, we propose that the positive dipole of the neighboring -helix backbone (G88-T89-T90-I91-T92-S93-K94) is responsible for retaining the cleaved phosphate. The revised catalytic mechanism involves a nucleophilic attack by a reactive water coordinated by both the T89 hydroxyl and a second water molecule coordinated directly by Mn2+. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII), containing the substrate fructose-1,6-bisphosphate (F1,6BP) bound, is presented in support of a novel catalytic mechanism for this class of enzymes.