PDBsum entry 2nom

Transferase

PDB id: 2nom

Contents

Protein chains

325 a.a. *

255 a.a. *

Ligands

DUT ×2

Metals

_MG ×2

Waters ×125

* Residue conservation analysis

PDB id:

2nom

Name:

Transferase

Title:

Terminal uridylyl transferase 4 from trypanosoma brucei with bound dutp

Structure:

RNA uridylyl transferase. Chain: a, b. Engineered: yes

Source:

Trypanosoma brucei. Organism_taxid: 5691. Gene: 841358, tb11.01.7300. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.40Å R-factor: 0.234 R-free: 0.301

Authors:

H.Luecke,J.Stagno

Key ref:

J.Stagno et al. (2007). UTP-bound and Apo Structures of a Minimal RNA Uridylyltransferase. J Mol Biol, 366, 882-899. PubMed id: 17189640 DOI: 10.1016/j.jmb.2006.11.065

Date:

25-Oct-06 Release date: 20-Feb-07

Protein chain

Q381M1  (Q381M1_TRYB2) -  Terminal uridylyltransferase 4 from Trypanosoma brucei brucei (strain 927/4 GUTat10.1)

Seq: 333 a.a.

Struc: 325 a.a.

Protein chain

Q381M1  (Q381M1_TRYB2) -  Terminal uridylyltransferase 4 from Trypanosoma brucei brucei (strain 927/4 GUTat10.1)

Seq: 333 a.a.

Struc: 255 a.a.

Enzyme reactions

• Enzyme class: Chains A, B: E.C.2.7.7.52 - Rna uridylyltransferase.
• Reaction: RNA(n) + UTP = RNA(n)-3'-uridine ribonucleotide + diphosphate

RNA(n) + UTP (Bound ligand (Het Group name = DUT) matches with 96.55% similarity) = RNA(n)-3'-uridine ribonucleotide + diphosphate

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

reference

DOI no: 10.1016/j.jmb.2006.11.065

J Mol Biol 366:882-899 (2007)

PubMed id: 17189640

UTP-bound and Apo Structures of a Minimal RNA Uridylyltransferase.

J.Stagno, I.Aphasizheva, A.Rosengarth, H.Luecke, R.Aphasizhev.

ABSTRACT

3'-Uridylylation of RNA is emerging as a phylogenetically widespread phenomenon involved in processing events as diverse as uridine insertion/deletion RNA editing in mitochondria of trypanosomes and small nuclear RNA (snRNA) maturation in humans. This reaction is catalyzed by terminal uridylyltransferases (TUTases), which are template-independent RNA nucleotidyltransferases that specifically recognize UTP and belong to a large enzyme superfamily typified by DNA polymerase beta. Multiple TUTases, recently identified in trypanosomes, as well as a U6 snRNA-specific TUTase enzyme in humans, are highly divergent at the protein sequence level. However, they all possess conserved catalytic and UTP recognition domains, often accompanied by various auxiliary modules present at the termini or between conserved domains. Here we report identification, structural and biochemical analyses of a novel trypanosomal TUTase, TbTUT4, which represents a minimal catalytically active RNA uridylyltransferase. The TbTUT4 consists of only two domains that define the catalytic center at the bottom of the nucleoside triphosphate and RNA substrate binding cleft. The 2.0 A crystal structure reveals two significantly different conformations of this TUTase: one molecule is in a relatively open apo conformation, whereas the other displays a more compact TUTase-UTP complex. A single nucleoside triphosphate is bound in the active site by a complex network of interactions between amino acid residues, a magnesium ion and highly ordered water molecules with the UTP's base, ribose and phosphate moieties. The structure-guided mutagenesis and cross-linking studies define the amino acids essential for catalysis, uracil base recognition, ribose binding and phosphate coordination by uridylyltransferases. In addition, the cluster of positively charged residues involved in RNA binding is identified. We also report a 2.4 A crystal structure of TbTUT4 with the bound 2' deoxyribonucleoside, which provides the structural basis of the enzyme's preference toward ribonucleotides.

Selected figure(s)

Figure 4.
Figure 4. Structural superposition of (a) TbRET2 (teal; PDB code 2B51) with TbTUT4 (red) with the middle domain, which is only present in TbRET2, on the left. (b) Superposition of ScPAP (gray; PDB code 1FA0) with TbTUT4 (red), with an additional domain at the C terminus, which is only present in ScPAP, on the upper right. UTP/Mg^2+ are included for TbTUT4 for visual reference. The Figure was generated using PyMOL [http://www.pymol.org]. Figure 4. Structural superposition of (a) TbRET2 (teal; PDB code 2B51) with TbTUT4 (red) with the middle domain, which is only present in TbRET2, on the left. (b) Superposition of ScPAP (gray; PDB code 1FA0) with TbTUT4 (red), with an additional domain at the C terminus, which is only present in ScPAP, on the upper right. UTP/Mg^2+ are included for TbTUT4 for visual reference. The Figure was generated using PyMOL [http://www.pymol.org].

Figure 6.
Figure 6. Key protein–UTP contacts in the UTP binding site. (a) UTP observed in molecule A shown with electron density from a composite annealed omit map contoured at 1.0σ. (b) Superposition of the active sites of TbRET2 (teal) and TbTUT4 (red). Residue labels are for TbTUT4. (c) Comparison of UTP binding via active site residues and their respective hydrogen bond networks for TbTUT4 and TbRET2. Selected water molecules (cyan spheres) were included to illustrate their significance in coordinating the uracil base while others were left out to improve clarity. The metal cations are shown as spheres: for TbTUT4 a Mg^2+ (magenta sphere), and for TbRET2 a Mn^2+ (black sphere). The Figurewas generated using PyMOL [http://www.pymol.org]. Figure 6. Key protein–UTP contacts in the UTP binding site. (a) UTP observed in molecule A shown with electron density from a composite annealed omit map contoured at 1.0σ. (b) Superposition of the active sites of TbRET2 (teal) and TbTUT4 (red). Residue labels are for TbTUT4. (c) Comparison of UTP binding via active site residues and their respective hydrogen bond networks for TbTUT4 and TbRET2. Selected water molecules (cyan spheres) were included to illustrate their significance in coordinating the uracil base while others were left out to improve clarity. The metal cations are shown as spheres: for TbTUT4 a Mg^2+ (magenta sphere), and for TbRET2 a Mn^2+ (black sphere). The Figurewas generated using PyMOL [http://www.pymol.org].

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 366, 882-899) copyright 2007.

Figures were selected by an automated process.