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 ␣-D-ribose 5-phosphate (ribose 5-phosphate) and ␣-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Enzyme-catalyzed phosphoryl transfer forms the basis for many biological, bioenergetic, and regulatory processes and is one of the most common cellular reactions (1). Numerous enzyme families have evolved mechanistically distinct solutions for phosphoryl transfer (2). Phosphomutases are phosphotransfer enzymes that rearrange the position of phosphate within a substrate molecule through either intramolecular (i.e. the phosphate is transferred to a different position on the same molecule) or intermolecular phosphoryl transfer (i.e. the phosphate is transferred from one substrate molecule to another).Bacterial phosphopentomutases (PPMs) 3 (EC 5.4.2.7) interconvert ribose 1-phosphate and ribose 5-phosphate, which bridges glucose metabolism and RNA biosynthesis (3). The importance of this reaction has recently been underscored by the observation that targeted deletion of the gene encoding PPM in the pathogen Francisella tularensis (deoB) results in markedly decreased virulence (4). PPMs appear to be biochemically and structurally distinct from their human congeners (5, 6), making them potential targets for antibiotic development.Sequence clustering classifies prokaryotic PPMs within the alkaline phosphatase superfamily of metalloenzymes, which includes a range of functionally diverse enzymes such as cofactor-independent phosphoglycerate mutase, phosphodiesterase, and estrone and aryl sulfatases (7). The majority of alkaline phosphatase superfamily enzymes catalyze a hydrolase reaction; however, both PPM (5) and the cofactor-independent phosphoglycerate mutase catalyze phosphomutase reactions (8, 9).All previously characterized alkaline phosphatase superfamily members follow a unified general reaction mechanism ( Fig. 1) (10). In alkaline phosphatase itself (11, 12), the catalytically competent enzyme has an unphosphorylated catalytic nucleophile, Ser-102 (Fig. 1, state 1). Turnover is initiated when the metallocenter activates a phosphoester donor substrate (R D -OPO 3 H Ϫ ) (Fig. 1, state 2) to transfer the phosphoryl group to the hydroxyl of Ser-102 (Fig. 1, state 3). This results in a covalent phosphoenzyme intermediate (E-OPO 3 H Ϫ ) (Fig. 1, state 4). A second phosphoryl transfer from the enzyme to the acceptor water molecule (Fig. 1, states 5 and 6) completes the reaction cycle. This general reaction mechanism has also been verified * This work was supported, in whole or in part, by National Institutes of Health Grants GM079419 (to T. M. I.), GM077189 (to B. O. B.), GM051366 (to B. E. W.), DK070787 (to B. E. W.), T32 NS07491 (to T. D. P.), T32 GM008320 (to T. D. P.), T90 DA022873 (to D. P. N.), and T32 GM07628 (to G. R. W.). This work was also supported by a pilot award funded by the Vanderbilt Instit...

Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39

“…6B]), are S -glutathionylated in cells. We show below that GapA S -glutathionylation is inhibitory; interestingly, DeoB S -glutathionylation may also be regulatory since the modified Cys is very close to the active site (67) (Table S2D). …”

Divergence and Convergence in Enzyme Evolution

“…In phosphopentomutase, the phosphorylated Thr residue appears to be present in the ground state, leading to the suggestion that the substrate enters this enzyme at a different point in the catalytic cycle than in AlkP (40). A phosphorylated Thr residue has also been reported in the active site of LtaS (38).…”

Molecular Differences between a Mutase and a Phosphatase: Investigations of the Activation Step in Bacillus cereus Phosphopentomutase

“…Indeed, the structure of B. cereus PPM is predominated by a core domain with a fold related to alkaline phosphatase; however, it also contains a cap domain that is unique to phosphopentomutases (Figure 1) (6). The active site is located at the interface between these two domains.…”

“…The active site is located at the interface between these two domains. Previous structure-based enzymology of B. cereus PPM (6) revealed numerous architectural and mechanistic parallels between PPM and alkaline phosphatase. The active sites of PPM and alkaline phosphatase exhibit virtually identical geometry with both containing a catalytic nucleophile (Thr-85 in B. cereus PPM and Ser-102 in Escherichia coli alkaline phosphatase (7)), and a dimetallo center (di-Mn 2+ in B. cereus PPM and di-Zn 2+ in E. coli alkaline phosphatase (8)).…”

“…The coordinating residues to the dimetallo center are absolutely conserved in both sequence and structure with the exception of an additional ligand within the B. cereus active site, Asp-156, that is located on the unique, cap domain (6, 9). Accordingly, the alkaline phosphatase reaction cycle was used as a starting point for developing a reaction mechanism specific for PPM (6). …”