Insights into the catalytic mechanisms of the protein enzymes, PFKFB3 and VldE, using x-ray crystallography

Abstract

This work describes the crystallographic studies of two enzymes and provides mechanistic insights into their respective catalytic processes. The first study investigates the molecular basis of the fructose-2,6-bisphosphatase reaction of the inducible isoform of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). The bifunctional enzyme is solely responsible for the cellular concentration of a regulator of glucose metabolism, fructose-2,6-bisphosphate. PFKB3 was investigated using the crystal structures of the enzyme in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF4), and in a complex with pyrophosphate (PFKFB3•PPi). With these structures, the structures of the Michaelis complex and the transition state were extrapolated. Additionally the C-terminal domain (residues 440-446) was rearranged in PFKFB3•PPi, implying that this domain plays a critical role in binding of substrate to, and release of product from, the bisphosphatase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. The second study investigates the molecular basis of the reaction catalyzed by the pseudo-glycosyltransferase, VldE. VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the anomeric configuration to form validoxylamine A 7´-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7´-phosphate, and in complex with GDP and trehalose. The structures of VldE with the catalytic site in both an “open” and “closed” conformation are also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA, could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the anomeric configuration of the donated cyclitol.