Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia coli D-3-Phosphoglycerate Dehydrogenase

D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC(50) values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC(50) value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced K(m), k(cat), and k(cat)/K(m). We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC(50) of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes.