Bdellovibrio bacteriovorus phosphoglucose isomerase structures reveal novel rigidity in the active site of a selected subset of enzymes upon substrate binding

Abstract

Glycolysis and gluconeogenesis are central pathways of metabolism across all domains of life. A prominent enzyme in these pathways is phosphoglucose isomerase (PGI), which mediates the interconversion of glucose-6-phosphate and fructose-6-phosphate. The predatory bacterium Bdellovibrio bacteriovorus leads a complex life cycle, switching between intraperiplasmic replicative and extracellular 'hunter' attack-phase stages. Passage through this complex life cycle involves different metabolic states. Here we present the unliganded and substrate-bound structures of the B. bacteriovorus PGI, solved to 1.74 Å and 1.67 Å, respectively. These structures reveal that an induced-fit conformational change within the active site is not a prerequisite for the binding of substrates in some PGIs. Crucially, we suggest a phenylalanine residue, conserved across most PGI enzymes but substituted for glycine in B. bacteriovorus and other select organisms, is central to the induced-fit mode of substrate recognition for PGIs. This enzyme also represents the smallest conventional PGI characterized to date and probably represents the minimal requirements for a functional PGI.

Keywords: Bdellovibrio bacteriovorus HD100; fructose-6-phosphate; glucose-6-phosphate; glycolysis; metabolism; phosphoglucose isomerase.

Figure 1.

Structure of BbPGI. (a) Domain boundaries and their corresponding position in BbPGI. LR, linker regions; CTS, C-terminal subdomain. (b) Orthogonal views of the BbPGI dimer assembly with dimer partners coloured blue and orange. The hook region which contributes to the dimeric arrangement is labelled. C-terminus and N-terminus are marked by C and N, respectively.

Figure 2.

The coordination of F6P in the active site. A F_o − F_c omit map indicating the presence of F6P (yellow) is depicted as a green mesh (contoured at 3σ). The ligand is coordinated by numerous hydrogen bonds represented by dashed lines. Peptide backbone displayed as sticks with opposing protomers of the dimer coloured in blue and orange.

Figure 3.

Tracking conformational changes in the active site. (a) rPGI (unliganded, purple) loops 209–215 and 245–259 move towards the active site upon F6P (green) or 5-phospho-d-arabinonate (cyan) binding to interact with the phosphoryl group (indicated by arrow). Binding of ligand also drives residues 385–389 towards the active site to re-position the catalytic histidine (H388). (b) Minimal backbone conformational movements between unliganded (orange) and ligand-bound (yellow) BbPGI. (c) Sequence alignment of BbPGI against PGIs of close relatives and model organisms. Numbered relative to BbPGI with red arrows indicating organisms with a Gly at the semi-conserved Phe position (green asterisk).