PDBsum entry 2ff1

Hydrolase

PDB id: 2ff1

Contents

Protein chains

314 a.a. *

Ligands

IMH ×2

Metals

_CA ×2

Waters ×378

* Residue conservation analysis

PDB id:

2ff1

Name:

Hydrolase

Title:

Crystal structure of trypanosoma vivax nucleoside hydrolase soaked with immucillinh

Structure:

Iag-nucleoside hydrolase. Chain: a, b. Fragment: iag-nh. Engineered: yes

Source:

Trypanosoma vivax. Organism_taxid: 5699. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.07Å R-factor: 0.178 R-free: 0.225

Authors:

W.Versees,J.Barlow,J.Steyaert

Key ref:

W.Versées et al. (2006). Transition-state complex of the purine-specific nucleoside hydrolase of T. vivax: enzyme conformational changes and implications for catalysis. J Mol Biol, 359, 331-346. PubMed id: 16630632 DOI: 10.1016/j.jmb.2006.03.026

Date:

18-Dec-05 Release date: 23-May-06

Protein chains

Q9GPQ4  (Q9GPQ4_TRYVI) -  IAG-nucleoside hydrolase from Trypanosoma vivax

Seq: 327 a.a.

Struc: 314 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.2.1 - purine nucleosidase.
• Reaction: a purine D-ribonucleoside + H2O = a purine nucleobase + D-ribose

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

Key reference

DOI no: 10.1016/j.jmb.2006.03.026

J Mol Biol 359:331-346 (2006)

PubMed id: 16630632

Transition-state complex of the purine-specific nucleoside hydrolase of T. vivax: enzyme conformational changes and implications for catalysis.

W.Versées, J.Barlow, J.Steyaert.

ABSTRACT

Nucleoside hydrolases cleave the N-glycosidic bond of ribonucleosides. Crystal structures of the purine-specific nucleoside hydrolase from Trypanosoma vivax have previously been solved in complex with inhibitors or a substrate. All these structures show the dimeric T. vivax nucleoside hydrolase with an "open" active site with a highly flexible loop (loop 2) in its vicinity. Here, we present the crystal structures of the T. vivax nucleoside hydrolase with both soaked (TvNH-ImmH(soak)) and co-crystallised (TvNH-ImmH(co)) transition-state inhibitor immucillin H (ImmH or (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol) to 2.1 A and 2.2 A resolution, respectively. In the co-crystallised structure, loop 2 is ordered and folds over the active site, establishing previously unobserved enzyme-inhibitor interactions. As such this structure presents the first complete picture of a purine-specific NH active site, including leaving group interactions. In the closed active site, a water channel of highly ordered water molecules leads out from the N7 of the nucleoside toward bulk solvent, while Trp260 approaches the nucleobase in a tight parallel stacking interaction. Together with mutagenesis results, this structure rules out a mechanism of leaving group activation by general acid catalysis, as proposed for base-aspecific nucleoside hydrolases. Instead, the structure is consistent with the previously proposed mechanism of leaving group protonation in the T. vivax nucleoside hydrolase where aromatic stacking with Trp260 and an intramolecular O5'-H8C hydrogen bond increase the pKa of the N7 sufficiently to allow protonation by solvent. A mechanism that couples loop closure to the positioning of active site residues is proposed based on a comparison of the soaked structure with the co-crystallized structure. Interestingly, the dimer interface area increases by 40% upon closure of loop 2, with loop 1 of one subunit interacting with loop 2 of the other subunit, suggesting a relationship between the dimeric form of the enzyme and its catalytic activity.

Selected figure(s)

Figure 3.
Figure 3. Comparison of the quaternary structure and subunit interface of TvNH-ImmH(soak) (a) and TvNH-ImmH(co) (b). Loop 1 is shown in blue, loop 2 in green. The Ca^2+ is shown as a grey sphere, ImmH is represented as a CPK model. The Figure was made with PyMOL.60

Figure 4.
Figure 4. (a) Active site of the closed TvNH-ImmH(co) structure. The residues Arg252 and Asp255 are provided by loop 2. The bound inhibitor, ImmH, is shown in yellow. The electron density, contoured at 3s, of an F[o] -F[c] simulated annealed omit map calculated without the inhibitor is also shown. The Ca^2+ and water molecules are represented as grey and red spheres, respectively. The "nucleophilic" water molecule, three water molecules forming a water channel from the N7 of the nucleobase toward bulk solvent, and a water molecule interacting with the N1 of the nucleobase are shown. The numbering of the B-subunit is used for the water molecules. The Figure was made with PyMOL.60 (b) Schematic representation of interactions in the active site of the TvNH-ImmH(co) structure. ImmH is shown in green. The color codes for the Ca^2+ and water molecules are the same as above.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 359, 331-346) copyright 2006.

Figures were selected by the author.