Structure and molecular mechanism of Bacillus anthracis cofactor-independent phosphoglycerate mutase: a crucial enzyme for spores and growing cells of Bacillus species

Abstract

Phosphoglycerate mutases (PGMs) catalyze the isomerization of 2- and 3-phosphoglycerates and are essential for glucose metabolism in most organisms. This study reports the production, structure, and molecular dynamics analysis of Bacillus anthracis cofactor-independent PGM (iPGM). The three-dimensional structure of B. anthracis PGM is composed of two structural and functional domains, the phosphatase and transferase. The structural relationship between these two domains is different than in the B. stearothermophilus iPGM structure determined previously. However, the structures of the two domains of B. anthracis iPGM show a high degree of similarity to those in B. stearothermophilus iPGM. The novel domain arrangement in B. anthracis iPGM and the dynamic property of these domains is directly linked to the mechanism of enzyme catalysis, in which substrate binding is proposed to result in close association of the two domains. The structure of B. anthracis iPGM and the molecular dynamics of this structure provide unique insight into the mechanism of iPGM catalysis, in particular the roles of changes in coordination geometry of the enzyme's two bivalent metal ions and the regulation of this enzyme's activity by changes in intracellular pH during spore formation and germination in Bacillus species.

FIGURE 1

Sequence analysis and production of B. anthracis iPGM. (a) Alignment of sequences of B. anthracis and B. stearothermophilus iPGMs. The overall sequence identity is 78%; however, in the phosphatase domain the identity increased to 79%, and for the transferase domain decreased to 74%. Conserved residues are marked by an asterisk, the residues of the catalytic site are marked by solid circles with the exception of catalytic Ser-61, which is marked by a solid triangle. (b) Electrophoretic analysis. Coomassie brilliant blue stained 10% SDS-PAGE gel: lane 1, protein molecular mass standards; lane 2, 3s mg of purified B. anthracis iPGM.

FIGURE 2

Three-dimensional structure of B. anthracis iPGM. (a) Overall ribbon representation of the molecule. This single peptide chain molecule assumes a two-domain structure with phosphatase and transferase domains. These domains are connected by two short peptide linkers, linker1 and 2. The structure is color coded by secondary structure elements: helices, red; β-sheets, yellow; and loops, green. Both N- and C-termini are labeled. The active site residues are shown in a ball and stick fashion (color-coded by element: carbon, green; oxygen, red; and nitrogen, blue). These residues belong to the two separate domains, phosphatase and transferase. For catalysis, these domains must move within reach of one another. The total of 13 amino acid residues take part in catalysis together with two essential Mn²⁺ ions, Mn¹ and Mn², and one water molecule, Wat. (b) Coordination spheres of catalytic Mn²⁺ ions. The coordination sphere of both the Mn²⁺ ions differs from that of distorted square pyramidal observed for B. stearothermophilus iPGM bound to PGA. For the B. anthracis iPGM without PGA, Mn¹ assumes a distorted square pyramidal coordination but the apex has moved from NE2 of His-461 to a different ligand. Mn² is in distorted octahedral coordination, altered from a distorted square pyramidal coordination, with the apex at OG of Ser-61. The atoms occupying the apex positions are NE2 of His-406 for Mn¹ (distorted square pyramidal coordination), and OD1 of Asp-11 and OD2 of Asp-443 for Mn² (distorted octahedral coordination). (c) Structure of catalytic site of B. stearothermophilus iPGM. This iPGM was crystallized with both domains close together with its catalytic residues in functional positions as during enzyme catalysis. A 3PGA substrate/product molecule is also shown. The residue numbering scheme follows that of B. anthracis iPGM enzyme plus one (i.e., B. anthracis Ser-61 is equivalent to B. stearothermophilus Ser-62). The coordination spheres of both Mn²⁺ ions were also depicted utilizing thin lines. The coordination geometry for both ions is distorted square pyramidal with NE2 of His-462 and OG of Ser-62 occupying the apex positions for Mn¹ and Mn², respectively (6).

FIGURE 3

Comparison of iPGM structures and their dynamic properties. (a) Structural differences between structures of iPGMs of Bacillus species illustrated by DynDom analysis. Two domains (red and blue) are connected by hinge regions colored green. The arrow represents the axis of rotation of the two domains relative to each other. The structural difference between the two structures may be represented as a near pure rotation about the green hinge residues with negligible independent translation. (b) Stereo view of a superposition between the open (dark blue; based on the B. anthracis iPGM structure) and closed (red; based on the B. stearothermophilus iPGM structure) crystal structures and the average structure obtained from the stable portion of the open trajectory (cyan). The structures were superimposed using Cα atoms of the phosphatase domain alone. It is clear that the significant divergence of the average trajectory structure (cyan) from the initial open structure (dark blue) was not in the direction of the closed structure (red). (c) Superposition between the open structure and its average trajectory structure rotated 90° with respect to the orientation in a. 2PGA is presented in green. The difference between the average open structure during MD and the starting open crystal structure corresponds to a twisting of the phosphatase and mutase domains.

FIGURE 4

Geometric analyses of the simulations of open B. anthracis and closed B. stearothermophilus iPGM structures. (a) Cα RMS differences to open and closed structures. RMS values calculated using all Cα atoms. The open, closed, and closed (ligand artificially removed) simulations are compared to their respective starting structures in the black, green, and cyan traces, respectively. These traces show that the closed structure is stable during the simulation and only marginally less so when its ligand has been removed. In contrast, the open simulation stabilizes at a conformation significantly different (RMSD of ∼0.5 nm) from the crystal structure. The upper traces, colored red, blue, and orange for the open, closed, and closed (ligand removed) (model structures) simulations show the cross comparisons of open simulations with closed structures and vice versa. They show that the fluctuations during the MD simulations do not cause the open simulations to more closely approximate the closed structures and vice versa. (b) Cα-Cα distances (nm) indicating the degree of closure of the catalytic site. For the simulation of closed B. stearothermophilus enzyme they are Lys-364 to Arg-153 (blue) or Lys-364 to Arg-264 (green). In the open B. anthracis simulation, the corresponding distances are Lys-363 to Arg-152 (black) and Lys-363 to Arg-263 (red). These distances measure the degree of closure of the catalytic site at the local level. They show that the catalytic site of the closed simulation remains closed throughout (green and blue traces), whereas the catalytic site of the open simulation is more conformationally variable but shows no tendency to close. (c) Additional Cα-Cα distances (nm) indicating the degree of closure of the catalytic site. The distances shown correspond to those depicted in panel b but measure the separation of the side-chain atoms Lys NZ and Arg CZ (nm) rather than the separation of their Cα atoms.