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Title: Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle

Abstract

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 Mn{sup 2+}-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-[{sup 18}O{sub 3}]phosphate and [U-{sup 13}C{sub 5}]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 two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

Authors:
; ; ; ; ; ; ;  [1]
  1. (Vanderbilt)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
UNIVERSITYNIH
OSTI Identifier:
1021786
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.
Additional Journal Information:
Journal Volume: 286; Journal Issue: (10) ; 03, 2011; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; ALKALINE PHOSPHATASE; BACILLUS CEREUS; CRYSTAL STRUCTURE; CRYSTALLOGRAPHY; ENZYME ACTIVITY; ENZYMES; GLUCOSE; MASS SPECTROSCOPY; METABOLISM; MIXTURES; PHOSPHATASES; PHOSPHORYLATION; PROTEINS; REACTION KINETICS; RESIDUES; RESOLUTION; RIBOSE; SUBSTRATES; TRANSFERASES

Citation Formats

Panosian, Timothy D., Nannemann, David P., Watkins, Guy R., Phelan, Vanessa V., McDonald, W. Hayes, Wadzinski, Brian E., Bachmann, Brian O., and Iverson, Tina M. Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle. United States: N. p., 2011. Web. doi:10.1074/jbc.M110.201350.
Panosian, Timothy D., Nannemann, David P., Watkins, Guy R., Phelan, Vanessa V., McDonald, W. Hayes, Wadzinski, Brian E., Bachmann, Brian O., & Iverson, Tina M. Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle. United States. doi:10.1074/jbc.M110.201350.
Panosian, Timothy D., Nannemann, David P., Watkins, Guy R., Phelan, Vanessa V., McDonald, W. Hayes, Wadzinski, Brian E., Bachmann, Brian O., and Iverson, Tina M. Thu . "Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle". United States. doi:10.1074/jbc.M110.201350.
@article{osti_1021786,
title = {Bacillus cereus Phosphopentomutase Is an Alkaline Phosphatase Family Member That Exhibits an Altered Entry Point into the Catalytic Cycle},
author = {Panosian, Timothy D. and Nannemann, David P. and Watkins, Guy R. and Phelan, Vanessa V. and McDonald, W. Hayes and Wadzinski, Brian E. and Bachmann, Brian O. and Iverson, Tina M.},
abstractNote = {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 Mn{sup 2+}-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-[{sup 18}O{sub 3}]phosphate and [U-{sup 13}C{sub 5}]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 two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.},
doi = {10.1074/jbc.M110.201350},
journal = {J. Biol. Chem.},
issn = {0021-9258},
number = (10) ; 03, 2011,
volume = 286,
place = {United States},
year = {2011},
month = {9}
}