Mapping of a Regulatory Site of the Escherichia coli ADP-Glucose Pyrophosphorylase.

Jaina A Bhayani,Miguel A Ballicora,Anisha Sharma,Benjamin L Hill,Alberto A Iglesias,Kenneth W Olsen

doi:10.3389/fmolb.2019.00089

Abstract

The enzyme ADP-glucose pyrophosphorylase (ADP-Glc PPase) controls the biosynthesis of glycogen in bacteria and starch in plants. It is regulated by various activators in different organisms according to their metabolic characteristics. In Escherichia coli, the major allosteric activator is fructose 1,6-bisphosphate (FBP). Other potent activator analogs include 1,6-hexanediol bisphosphate (HBP) and pyridoxal 5′-phosphate (PLP). Recently, a crystal structure with FBP bound was reported (PDB ID: 5L6S). However, it is possible that the FBP site found is not directly responsible for the activation of the enzyme. We hypothesized FBP activates by binding one of its phosphate groups to another site (“P1”) in which a sulfate molecule was observed. In the E. coli enzyme, Arg40, Arg52, and Arg386 are part of this “P1” pocket and tightly complex this sulfate, which is also present in the crystal structures of ADP-Glc PPases from Agrobacterium tumefaciens and Solanum tuberosum. To test this hypothesis, we modeled alternative binding conformations of FBP, HBP, and PLP into “P1.” In addition, we performed a scanning mutagenesis of Arg residues near potential phosphate binding sites (“P1,” “P2,” “P3”). We found that Arg40 and Arg52 are essential for FBP and PLP binding and activation. In addition, mutation of Arg386 to Ala decreased the apparent affinity for the activators more than 35-fold. We propose that the activator binds at this “P1” pocket, as well as “P2.” Arg40 and Arg52 are highly conserved residues and they may be a common feature to complex the phosphate moiety of different sugar phosphate activators in the ADP-Glc PPase family.

Highlights

ADP-glucose pyrophosphorylase (EC 2.7.7.27; ADP-Glc PPase) is a regulatory, homotetrameric enzyme that catalyzes the reaction between adenosine triphosphate (ATP) and glucose 1-phosphate (Glc1P), producing ADP-glucose (ADP-Glc) and pyrophosphate (PPi) (Figure 1) (Ballicora et al, 2003)
E. coli ADP-Glc PPase was categorized as an enzyme solely activated by FBP (Ballicora et al, 2003), but it was recently found that pyruvate is a secondary activator that synergistically enhances the effect of FBP (Asencion Diez et al, 2014)
We hypothesized that the FBP binding site involved in the activation of E. coli ADPGlc PPase is partially located where a sulfate has been detected in the crystal structure (Cifuente et al, 2016)

Summary

INTRODUCTION

ADP-glucose pyrophosphorylase (EC 2.7.7.27; ADP-Glc PPase) is a regulatory, homotetrameric enzyme that catalyzes the reaction between adenosine triphosphate (ATP) and glucose 1-phosphate (Glc1P), producing ADP-glucose (ADP-Glc) and pyrophosphate (PPi) (Figure 1) (Ballicora et al, 2003). Mutagenesis in the A. tumefaciens enzyme indicated that the pyruvate regulatory site does not completely overlap with the Fru6P site (Gomez-Casati et al, 2001) This was in agreement with the structures of the enzymes from E. coli and A. tumefaciens with activators bound (FBP and pyruvate, respectively) (Cifuente et al, 2016; Hill et al, 2019). The crystal structure of ADP-Glc PPase from E. coli was solved in the presence of the β-D-furanose form of FBP (PDB ID: 5L6S) (Cifuente et al, 2016) In this structure, the phosphates from FBP are interacting with Lys, Arg130, and residues from the C-terminal domain (Arg419, Arg423, and backbone interactions with Gln429 and Glu430). Structural, binding, and kinetic analysis allowed us to conclude there is a site, occupied by a sulfate in the crystal structure, which is critical for activation of the enzyme

MATERIALS AND METHODS

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DATA AVAILABILITY STATEMENT