CryoEM structure of the soluble region of the MS-ring.

(A) Map of the C33 RBM3 colored by the local resolution, which ranges from 2.6 Å in blue to 3.4 Å in red. Regions marked with the magenta boxes are shown in panel B with corresponding density. (B) Representative fit of models into the cryoEM density map.</p

Interdomain flexibility between the β-collar and RBM3.

(A) Superposition of the β-collar from this study (red) superimposed with the equivalent region in the coordinates deposited by Johnson et al (cyan, PDB:6SCN [17]) highlights a 5.7° difference in angle between the β-collar and RBM3, as calculated using DynDom [37]. This translates into a shift of position of RBM3 of 5.4 Å and a ~10 Å difference in diameter of the MS-ring, even with the same number of protomers. (B) The outer diameter (measured from chain A, Leu-402 to chain Q, Leu-402) and inner diameter (measured from chain A, Leu-298 to chain Q, Leu-298) of the MS-ring structure finds an outer and inner diameter of our structure of 242 Å and 104 Å, respectively. (C) Using equivalent residues for the measurement, the outer and inner diameter of the MS-ring structure from PDB:6SCN [17] is 232 Å and 99 Å, respectively.</p

Domains of the MS-ring.

(A) Map of MS-ring colored by domain with the ß-collar region in orange, RBM3 in blue, RBM2 in yellow, RBM2outer-RBM1 in cyan, and membrane region in magenta (B) Separation of the individual domains of the MS-ring highlights the different resolutions and symmetries of each region of the post-assembly MS-ring.</p

Molecular Differences between a Mutase and a Phosphatase: Investigations of the Activation Step in <i>Bacillus cereus</i> 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 <i>Bacillus cereus</i> PPM suggested adaptations of the conserved alkaline phosphatase catalytic cycle. Notably, <i>B. cereus</i> 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 <i>B. cereus</i> 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