PDBsum entry: 1tmj

Transferase

PDB id: 1tmj

Contents

Protein chain

158 a.a. *

Metals

_CL

_MG ×2

Waters ×252

* Residue conservation analysis

PDB id:

1tmj

Name:

Transferase

Title:

Crystal structure of e.Coli apo-hppk(w89a) at 1.45 angstrom resolution

Structure:

2-amino-4-hydroxy-6- hydroxymethyldihydropteridine pyrophosphokinase. Chain: a. Synonym: 7,8-dihydro-6-hydroxymethylpterin- pyrophosphokinase, hppk, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, pppk. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: folk, b0142. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.45Å R-factor: 0.155 R-free: 0.174

Authors:

J.Blaszczyk,X.Ji

Key ref:

Y.Li et al. (2005). 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. PubMed id: 15952765 DOI: 10.1021/bi0503495

Date:

10-Jun-04 Release date: 21-Jun-05

Protein chain

P26281  (HPPK_ECOLI) -  2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase

Seq: 159 a.a.

Struc: 158 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.7.6.3 - 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase.
• Pathway: Folate Biosynthesis (late stages)
• Reaction: ATP + 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine = AMP + (2-amino-4-hydroxy-7,8-dihydropteridin-6-yl)methyl diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: folic acid and derivative biosynthetic process (2 terms)
• Biochemical function: nucleotide binding (5 terms)

reference

DOI no: 10.1021/bi0503495

Biochemistry 44:8590-8599 (2005)

PubMed id: 15952765

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.

Y.Li, J.Blaszczyk, Y.Wu, G.Shi, X.Ji, H.Yan.

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.