1TMJ

Crystal structure of E.coli apo-HPPK(W89A) at 1.45 Angstrom resolution

Summary for 1TMJ

• Entry DOI: 10.2210/pdb1tmj/pdb
• Related: 1CBK 1DY3 1EQ0 1EQM 1EX8 1F9H 1F9Y 1G4C 1HKA 1HQ2 1IM6 1KBR 1Q0N 1RB0 1RTZ 1RU1 1RU2 1TMM
• Descriptor: 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase, MAGNESIUM ION, CHLORIDE ION, ... (4 entities in total)
• Functional Keywords: pyrophosphokinase, pyrophosphoryl transfer, folate, hppk, 6-hydroxymethylpterin, 6-hydroxymethyl-7, 8-dihydropterin, antimicrobial agent, drug design, point mutant, structural mutagenesis, transferase
• Biological source: Escherichia coli
• Total number of polymer chains: 1
• Total formula weight: 17935.47

Authors

Blaszczyk, J.,Ji, X. (deposition date: 2004-06-10, release date: 2005-06-21, Last modification date: 2023-08-30)

Primary citation

Li, Y.,Blaszczyk, J.,Wu, Y.,Shi, G.,Ji, X.,Yan, H.
Is the Critical Role of Loop 3 of Escherichia coli 6-Hydroxymethyl-7,8-dihydropterin Pyrophosphokinase in Catalysis Due to Loop-3 Residues Arginine-84 and Tryptophan-89? Site-Directed Mutagenesis, Biochemical, and Crystallographic Studies.
Biochemistry, 44:8590-8599, 2005

PubMed Abstract: Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.

PubMed: 15952765
DOI: 10.1021/bi0503495

Experimental method

X-RAY DIFFRACTION (1.45 Å)