PDBsum entry: 1i0l

Transferase

PDB id: 1i0l

Contents

Protein chains

192 a.a. *

Ligands

7HP ×2

PRP ×2

Metals

_MG ×3

Waters ×159

* Residue conservation analysis

PDB id:

1i0l

Name:

Transferase

Title:

Analysis of an invariant aspartic acid in hprts-asparagine mutant

Structure:

Hypoxanthine-guanine phosphoribosyltransferase. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Trypanosoma cruzi. Organism_taxid: 5693. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PQS)

Resolution:

1.72Å R-factor: 0.210 R-free: 0.267

Authors:

B.Canyuk,P.J.Focia,A.E.Eakin

Key ref:

B.Canyuk et al. (2001). The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography. Biochemistry, 40, 2754-2765. PubMed id: 11258886 DOI: 10.1021/bi001195q

Date:

29-Jan-01 Release date: 29-May-02

Protein chains

Q27796  (Q27796_TRYCR) -  Hypoxanthine-guanine phosphoribosyltransferase

Seq: 221 a.a.

Struc: 192 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.4.2.8 - Hypoxanthine phosphoribosyltransferase.
• Reaction: IMP + diphosphate = hypoxanthine + 5-phospho-alpha-D-ribose 1-diphosphate

IMP + diphosphate = hypoxanthine (Bound ligand (Het Group name = 7HP) matches with 66.00% similarity) + 5-phospho-alpha-D-ribose 1-diphosphate (Bound ligand (Het Group name = PRP) corresponds exactly)

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

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (1 term)
• Biological process: nucleoside metabolic process (2 terms)
• Biochemical function: transferase activity (3 terms)

reference

DOI no: 10.1021/bi001195q

Biochemistry 40:2754-2765 (2001)

PubMed id: 11258886

The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography.

B.Canyuk, P.J.Focia, A.E.Eakin.

ABSTRACT

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.