Molecular Architecture of the Glucose 1-Phosphate Site in ADP-glucose Pyrophosphorylases

Jack Preiss,Clarisa Maria Bejar,Miguel Angel Ballicora,Xiangshu Jin

doi:10.1074/jbc.m607088200

Abstract

ADP-Glc pyrophosphorylase (PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP-Glc has been generated to study the substrate-binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the glucose moiety of the ADP-Glc ligand. The role of these amino acids (Glu(194), Ser(212), Tyr(216), Asp(239), Phe(240), Trp(274), and Asp(276)) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP-Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDP-Glc PPase. Therefore, the data reported here may have implications for other members of the nucleotide-diphosphoglucose PPase family.

Highlights

Most ADP-Glc PPases are allosterically regulated by small effector molecules
Homology Modeling—We obtained a three-dimensional model of E. coli ADP-Glc PPase by comparative modeling using the coordinates of the recently solved crystal structure of the potato tuber ADP-Glc PPase small subunit (Protein Data Bank code 1YP2) as a template as described under “Experimental Procedures” (Fig. 1A)
We have reported the first detailed characterization of the sugar phosphate site and the three-dimensional structure of E. coli ADP-Glc PPase, the Glc-1-P site

Summary

Introduction

Most ADP-Glc PPases are allosterically regulated by small effector molecules. these vary according to the source, they are all intermediates of the principal carbon assimilation pathway in the respective cell [2,3,4,5,6]. The enzymes from heterotrophic bacteria such as Escherichia coli are regulated by intermediates of the glycolytic pathway, with Fru-1,6-P2 as the main activator and AMP as the main inhibitor On another hand, the ADP-Glc PPases from cells performing oxygenic photosynthesis and assimilating atmospheric CO2 through the reductive pentose phosphate pathway or the Calvin cycle ( cyanobacteria, green algae, and photosynthetic tissues from higher plants) are activated by 3-phosphoglycerate and inhibited by Pi [6]. The first ADP-Glc PPase crystal structure became recently available when Jin et al [14] solved that of the homotetrameric Solanum tuberosum (potato tuber) small subunit in its allosterically inhibited form at a resolution of 2.1 Å They reported the structural determination of the enzyme complexed with either ATP or ADP-Glc at 2.6 and 2.2 Å, respectively. Results with Hex-1-P analogs, which differ from Glc-1-P in their hydroxyl groups, suggest that other residues in the active site participate in substrate binding

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