Related Experiment Video
Updated: May 25, 2026
Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus
T M Iverson1, Timothy D Panosian, William R Birmingham
1Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States. tina.iverson@vanderbilt.edu
Abstract:
Prokaryotic phosphopentomutases (PPMs) are di-Mn(2+) 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 PPM(T85E) phosphomimetic variant and the neutral corollary PPM(T85Q) determined that the side chain of Lys-240 underwent a change in conformation in response to active site charge, which modestly influenced the affinity for the small molecule activator α-D-glucose 1,6-bisphosphate. More strikingly, the structure of unphosphorylated B. cereus PPM revealed a dramatic change in the interdomain angle and a new hydrogen bonding interaction between the side chain of Asp-156 and the active site nucleophile, Thr-85. This hydrogen bonding interaction is predicted to align and activate Thr-85 for nucleophilic addition to α-D-glucose 1,6-bisphosphate, favoring the observed equilibrium phosphorylated state. Indeed, phosphorylation of Thr-85 is severely impaired in the PPM(D156A) variant even under stringent activation conditions. These results permit a proposal for activation of PPM and explain some of the essential features that distinguish between the catalytic cycles of PPM and alkaline phosphatase.
Insights
Prokaryotic phosphopentomutases (PPMs) are enzymes that interconvert ribose phosphates. This study reveals how PPMs activate their active site nucleophile, Thr-85, through a unique hydrogen bond, distinguishing their mechanism from alkaline phosphatases.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Prokaryotic phosphopentomutases (PPMs) are di-Mn(2+) enzymes catalyzing α-D-ribose 5-phosphate and α-D-ribose 1-phosphate interconversion.
- PPMs belong to the alkaline phosphatase superfamily but exhibit distinct catalytic mechanisms.
- Bacillus cereus PPM engages substrates with a phosphorylated active site nucleophile (Thr-85), unlike alkaline phosphatase.
Purpose of the Study:
- To elucidate the mechanistic divergence between prokaryotic phosphopentomutases (PPMs) and alkaline phosphatases.
- To understand the structural and biochemical basis for the unique substrate engagement and phosphoenzyme stability in B. cereus PPM.
Main Methods:
- Crystallographic and biochemical investigations.
- Analysis of PPM(T85E) phosphomimetic and PPM(T85Q) variants.
- Structural determination of unphosphorylated B. cereus PPM.
Main Results:
- Lys-240 conformation changed in response to active site charge, affecting activator affinity.
- Unphosphorylated B. cereus PPM structure showed altered interdomain angle and a novel Asp-156 to Thr-85 hydrogen bond.
- This hydrogen bond is predicted to activate Thr-85 for phosphorylation, and its disruption in PPM(D156A) impaired Thr-85 phosphorylation.
Conclusions:
- A mechanism for PPM activation involving a key hydrogen bond between Asp-156 and Thr-85 is proposed.
- This interaction explains the distinct catalytic cycle features differentiating PPMs from alkaline phosphatases.
Related Concept Videos
02:54Protein Kinases and Phosphatases
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
01:02Phosphorylation
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...