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

Clarisa Maria Bejar, Xiangshu Jin, Miguel Angel Ballicora, Jack Preiss
2006 Journal of Biological Chemistry  
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
more » ... ids (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. The biosynthetic pathways of starch and bacterial glycogen are very similar (1). The initial and key regulatory step is the formation of the glucosyl donor molecule ADP-Glc from ATP and Glc-1-P via a reaction catalyzed by ADP-glucose pyrophosphorylase (PPase 4 ; glucose-1-phosphate adenylyl-Downloaded from FIGURE 5. Superposition of the amino acids in the Glc-1-P site from three NDP-Glc PPases. A, superposition of residues from the E. coli ADP-Glc PPase model with carbons in white (this work) and from the crystal structures of P. aeruginosa RmlA (Protein Data Bank code 1G23) with carbons in magenta (18) and S. typhi CDP-Glc PPase (Protein Data Bank code 1TZF) with carbons in cyan (19). The ADP-Glc PPase model has root mean square deviations of 1.9 Å with RmlA and 2.2 Å with CDP-Glc PPase. We show the CDP-Glc PPase active site as it is in the active enzyme, with Asp 131 , Trp 232 , and Asp 236 belonging to one subunit and Glu 178 Ј and Lys 179 Ј from the neighboring monomer. The ADP-Glc molecule was modeled in ADP-Glc PPase, and Mg 2ϩ is present in the CDP-Glc PPase crystal structure. B, Ala 140 , Ser 212 , and Tyr 216 in E. coli ADP-Glc PPase (white carbons) overlaid with the homologous residues Tyr 129 , Asn 188 , and Phe 192 in CDP-Glc PPase (cyan carbons) and Leu 108 , Val 172 , and Tyr 176 in RmlA (magenta carbons). C, Asp 239 and Phe 240 in E. coli ADP-Glc PPase (white carbons) overlaid with the homologous residues Thr 208 and Trp 209 in CDP-Glc PPase (cyan carbons) and Glu 198 and Ile 199 in RmlA (magenta carbons).
doi:10.1074/jbc.m607088200 pmid:17079236 fatcat:54li3my5bneppc4gf7qh6vg6f4