Molecular differences between a mutase and a phosphatase: Investigations of the activation step in Bacillus cereus phosphopentomutase

Prokaryotic phosphopentomutases (PPMs) are di-Mn2+ enzymes that catalyze the interconversion of α-d-ribose 5-phosphate and α-d-ribose 1-phosphate at an active site located between two independently folded domains. These prokaryotic PPMs belong to the alkaline phosphatase superfamily, but previous studies of Bacillus cereus PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, B. cereus PPM engages substrates when the active site nucleophile, Thr-85, is phosphorylated. Further, the phosphoenzyme is stable throughout purification and crystallization. In contrast, alkaline phosphatase engages substrates when the active site nucleophile is dephosphorylated, and the phosphoenzyme reaction intermediate is only stably trapped in a catalytically compromised enzyme. Studies were undertaken to understand the divergence of these mechanisms. Crystallographic and biochemical investigations of the PPMT85E phosphomimetic variant and the neutral corollary PPMT85Q determined that the si...