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Publication Abstract from PubMed

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert alpha-D-ribose-5-phosphate (ribose-5-phosphate) and alpha-D-ribose-1-phosphate (ribose-1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high-resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose-5-phosphate and the activator alpha-D-glucose-1,6-bisphosphate (glucose-1,6-bisphosphate), and demonstrated that glucose-1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn2+-dependent enzyme turnover and demonstrated that glucose-1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose-5-[18O3]-phosphate and 13C5-ribose-5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the mechanism may be reconciled with intermolecular transfer if substrate encounters the enzyme at a different point in the catalytic cycle.

The Bacillus cereus phosphopentomutase is an alkaline phosphatase family member that exhibits an altered entry point into the catalytic cycle.,Panosian TD, Nannemann DP, Watkins G, Phelan VV, McDonald WH, Wadzinski BE, Bachmann BO, Iverson TM J Biol Chem. 2010 Dec 30. PMID:21193409^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.