Molecular insight into 2-phosphoglycolate activation of the phosphatase activity of bisphosphoglycerate mutase

Abstract

Bisphosphoglycerate mutase (BPGM) is an erythrocyte-specific multifunctional enzyme that is responsible for the regulation of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells through its synthase and phosphatase activities; the latter enzymatic function is stimulated by the endogenous activator 2-phosphoglycolate (2-PG). 2,3-BPG is a natural allosteric effector of hemoglobin (Hb) that is responsible for decreasing the affinity of Hb for oxygen to facilitate tissue oxygenation. Here, crystal structures of BPGM with 2-PG in the presence and absence of 3-phosphoglycerate are reported at 2.25 and 2.48 Å resolution, respectively. Structure analysis revealed a new binding site for 2-PG at the dimer interface for the first time, in addition to the expected active-site binding. Also, conformational non-equivalence of the two active sites was observed as one of the sites was found in an open conformation, with the residues at the active-site entrance, including Arg100, Arg116 and Arg117, and the C-terminus disordered. The kinetic result is consistent with the binding of 2-PG to an allosteric or noncatalytic site as well as the active site. This study paves the way for the rational targeting of BPGM for therapeutic purposes, especially for the treatment of sickle cell disease.

Characterization of the Escherichia coli pyridoxal 5′‐phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability

“…A sedimentation velocity (SV) experiment using absorbance and interference detectors was carried out using a Proteome Lab XL‐I analytical ultracentrifuge (AUC; Beckman Coulter) as per Aljahdali et al 41 YggS protein (1.5 mg) was loaded into AUC cell assemblies in PBS, pH 7.4 and centrifuged at 40,000 rpm, for 9 hr at 20°C. The resulting SV data was analyzed using the continuous c(s) module in SEDFIT version 16.36 as described in Schuck et al 42 …”

Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway

“…A sedimentation velocity (SV) experiment using absorbance and interference detectors was carried out using a Proteome Lab XL‐I analytical ultracentrifuge (Beckman Coulter, Brea, CA, USA) as previously described [47]. PdxI (1.5 mg) in 0.1 m Tris–HCl and 0.15 m NaCl was loaded into AUC cell assemblies in PBS, pH 7.4 and centrifuged at 116 480 g , for 9 h at 20 °C.…”

Metabolic Reprogramming in Sickle Cell Diseases: Pathophysiology and Drug Discovery Opportunities

“…Bisphosphoglycerate mutase (BPGM) is the central enzyme in the Rapoport-Leubering pathway, exclusively expressed in erythrocytes and placental cells [73,74]. BPGM regulates the intraerythrocytic level of 2,3-BPG by catalyzing both its synthesis and degradation [47,75]. The main activity of the enzyme is its synthase activity, which catalyzes the generation of 2,3-BPG from 1,3-BPG, an intermediate in glycolysis (Reaction 1; Figure 5).…”

“…While there are no known synthetic modulators of BPGM, its uniqueness to erythrocytes and its central role in 2,3-BPG production positions BPGM as an ideal target for SCD therapeutics. BPGM is a homodimer, with each monomer composed of two domains that are formed by six β-strands (named βA-F) and ten α-helices (named α1-10) with a molecular weight of 30 kDa per monomer [75,78]. The dimer is formed between the surface of the βC strands and α3 helices of the two monomers (PDB: 7n3r) (Figure 6).…”

“…Additionally, the active site of BPGM is highly charged, thus posing a problem for designing synthetic modulators that are equally potent and bioavailable. In a recent published study, our group identified a novel binding site at the dimer interface of BPGM, which when bound with ligand appears to affect the catalytic activity of the enzyme [75]. This new site could be targeted for designing BPGM modulators, either to activate the phosphatase activity or to inhibit the synthase activity to reduce 2,3-BPG levels in RBCs.…”