Mechanism of Catalysis of the Cofactor-independent Phosphoglycerate Mutase from Bacillus stearothermophilus

Abstract

The structure of the complex between the 2,3-diphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus and its 3-phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-phosphoglycerate and two Mn 2؉ ions at 1.7-Å resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2-or 3-phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase. Phosphoglycerate mutases (PGMs)1 catalyze three types of reactions including interconversion of 1,3-phosphoglycerate and 2,3-phosphoglycerate (23PGA) and of 3-phosphoglycerate (3PGA) and 2-phosphoglycerate (2PGA) as well as synthesis of 3PGA from 23PGA (1). There are two distinct types of PGMs, bisphosphoglycerate mutase and monophosphoglycerate mutase, and only the first type has the ability to perform all of the reactions listed above, while monophosphoglycerate mutases catalyze primarily the interconversion of 3PGA and 2PGA in both glycolysis and gluconeogenesis. There are also two classes of monophosphoglycerate mutases that are distinguished by their requirement for 23PGA for catalysis (1, 2). The PGMs requiring 23PGA for catalysis are termed 23PGA-dependent and are the predominant PGM in mammals, yeast, and some bacteria. They also have the ability to perform all reactions noted above but at significantly different rates. The monophosphoglycerate mutases that do not require DPG for catalysis are termed 23PGA-independent (iPGMs) and are the predominant PGM in plants and some other bacteria, including endosporeforming Gram-positive bacteria and their close relatives; iPGMs, unlike cofactor-dependent phosphoglycerate mutases and bisphosphoglycerate mutases, can only carry out the interconversion of 2PGA and 3PGA (1, 3, 4). The two classes of monophosphoglycerate mutases are extremely different in amino acid sequence, catalytic mechanism, and structure, both tertiary and quaternary. Despite the differences between these two types of PGMs, the iPGMs all have very conserved amino acid sequences, as do the cofactor-d...

The Serine/Threonine/Tyrosine Phosphoproteome of the Model Bacterium Bacillus subtilis

“…For some glycolytic enzymes phosphorylation on serine has been shown to occur as an intermediate step in catalysis. The best studied example is the phosphoglycerate mutase from Bacillus stearothermophilus where it was shown that reversible conversion of 2-to 3-phosphoglycerate occurs via formation of a phosphoenzyme intermediate phosphorylated at Ser 62 (52). Here we detected the same site occupied in the highly homologous phosphoglycerate mutase of B. subtilis.…”

Characterization of the cofactor‐independent phosphoglycerate mutase from Leishmania mexicana mexicana

“…Data concerning other active site histidines, His79 and His496, were ambiguous since the former could not be distinguished from His60 and the modification of the latter could not be confirmed by MS/MS analysis. Interestingly, Lys357 was not modified in spite of being a presumably highly nucleophilic residue, possibly able to withdraw a proton from Ser75, which corresponds to Ser62 in the B. stearothermophilus enzyme, the phosphorylated residue according to the proposed catalytic mechanism that involves a phosphoenzyme intermediate [35][36][37]. Previous site-directed mutagenesis studies on the castor plant i-PGAM have shown that the conserved histidines corresponding to LmPGAM His136 and His496 were both essential for catalytic activity, while mutating His467 rendered the enzyme insoluble [33].…”

“…It is indicated in the table which of these residues were modified or protected in the experiments with DEPC and substrate + DEPC. The location of the residues with respect to the active site or the surface was identified by sequence alignment with the B. stearothermophilus enzyme, of which the crystal structure is known [35,36] and by examining a recently solved, unpublished structure of LmPGAM (B. Poonperm, M. Walkinshaw and L. A. Fothergill-Gilmore, unpublished data). Figure 6 shows the spatial distribution of all conserved histidines, together with two important activesite residues, Lys357 and Ser75.…”

Analysis of the Function of a Putative 2,3-Diphosphoglyceric Acid-Dependent Phosphoglycerate Mutase from Bacillus subtilis

“…An alternative possibility is that, while the protein encoded by yhfR may not catalyze PGM activity, it may catalyze some similar reaction; for example, it appears possible that, with only minor changes, a dPGM could evolve into a phosphatase (1,19). Indeed, the structure of the active sites of iPGMs is almost identical to that of E. coli alkaline phosphatase (9). While further work will be required to definitively assess possible enzymatic reactions catalyzed by YhfR, this enzyme is clearly not needed for normal growth and differentiation of B. subtilis.…”

“…Two types of PGM have been identified; one is dependent on 2,3-diphosphoglycerate (DPG) for activity (dPGM), and the other is not (iPGM) (5,6). These two types of PGM differ strikingly in their structures, mechanisms, and amino acid sequences (3,5,6,8,9,10). Some organisms appear to contain only a single type of PGM, while others contain both types of PGM (2,5,6,7).…”