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

Qian Wang,Yifei Qi,Ning Yin,Luhua Lai,Andrea Cavalli

doi:10.1371/journal.pone.0094829

Qian Wang, Yifei Qi + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0094829

Copy DOI

Abstract

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 IC50 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 AC50 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 Km, kcat, and kcat/Km. 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 IC50 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.

Highlights

D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) catalyzes the first critical step in de novo L-serine biosynthesis, facilitating the transition of 3-phosphoglycerate (3-PG) into 3phosphohydroxypyruvate with NAD+ as a cofactor [1]
We have discovered three novel allosteric effectors and seven of their analogs for PGDH based on the predicted allosteric site I
Though the activities of compounds 1 and 2 were comparable to the endogenous allosteric effector, L-serine [33], the large size of site I is more suitable for further compound optimization and the discovery of novel allosteric effectors with diverse chemical structures

Summary

Introduction

D-3-phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) catalyzes the first critical step in de novo L-serine biosynthesis, facilitating the transition of 3-phosphoglycerate (3-PG) into 3phosphohydroxypyruvate (pPYR) with NAD+ as a cofactor [1]. It can be allosterically inhibited by serine, the end product of the pathway [2]. Each formed by a contact of the nucleotide-binding domain, further dimerize through contacts of the regulatory domains. It has been suggested that the binding of serine stabilizes the regulatory domain interface contacts and inhibits enzyme activity by limiting the movement of the rigid domains through flexible hinges, preventing the active sites from closing [4]

Methods

Results

Conclusion