PDBsum entry 3h0r

Ligase

PDB id: 3h0r

Contents

Protein chains

(+ 2 more) 478 a.a.

(+ 2 more) 410 a.a.

(+ 2 more) 91 a.a.

Ligands

ASN ×8

ADP ×2

ATP ×6

ASP ×2

Metals

_MN ×16

_ZN ×8

Waters ×49

References listed in PDB file

Key reference

Title

Insights into tRNA-Dependent amidotransferase evolution and catalysis from the structure of the aquifex aeolicus enzyme.

Authors

J.Wu, W.Bu, K.Sheppard, M.Kitabatake, S.T.Kwon, D.Söll, J.L.Smith.

Ref.

J Mol Biol, 2009, 391, 703-716. [DOI no: 10.1016/j.jmb.2009.06.014]

PubMed id

19520089

Abstract

Many bacteria form Gln-tRNA(Gln) and Asn-tRNA(Asn) by conversion of the misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) species catalyzed by the GatCAB amidotransferase in the presence of ATP and an amide donor (glutamine or asparagine). Here, we report the crystal structures of GatCAB from the hyperthermophilic bacterium Aquifex aeolicus, complexed with glutamine, asparagine, aspartate, ADP, or ATP. In contrast to the Staphylococcus aureus GatCAB, the A. aeolicus enzyme formed acyl-enzyme intermediates with either glutamine or asparagine, in line with the equally facile use by the amidotransferase of these amino acids as amide donors in the transamidation reaction. A water-filled ammonia channel is open throughout the length of the A. aeolicus GatCAB from the GatA active site to the synthetase catalytic pocket in the B-subunit. A non-catalytic Zn(2+) site in the A. aeolicus GatB stabilizes subunit contacts and the ammonia channel. Judged from sequence conservation in the known GatCAB sequences, the Zn(2+) binding motif was likely present in the primordial GatB/E, but became lost in certain lineages (e.g., S. aureus GatB). Two divalent metal binding sites, one permanent and the other transient, are present in the catalytic pocket of the A. aeolicus GatB. The two sites enable GatCAB to first phosphorylate the misacylated tRNA substrate and then amidate the activated intermediate to form the cognate products, Gln-tRNA(Gln) or Asn-tRNA(Asn).

Figure 3.
Fig. 3. Comparison of amidase active sites. (a) A-subunit of A. aeolicus GatCAB showing the acyl-enzyme intermediate of substrate Gln with Ser171. (b) Active site of MAE2 in complex with product malonate.^23 Arg158 in the amidase core region interacts with a carboxyl group of malonate (Mal). (c) Active site of FAAH with the inactivator methoxy arachidonyl phosphonate (MAP).^26 The phosphonate of the covalent adduct at nucleophilic Ser241 mimics the tetrahedral intermediate of the hydrolytic reaction. Aromatic and aliphatic residues in the substrate binding pocket are indicated. (d) Active site of PAM in complex with the inhibitor chymostatin (CST).^25 For each enzyme, the amidase core region (residues 62–192 of GatA, residues 52–176 of MAE2, residues 132–262 of FAAH, and residues 113–246 of PAM) is colored blue, and residues outside the core region are colored green. Residues in the Ser–cisSer–Lys catalytic scissors of each enzyme and those interacting with ligands are shown as thin sticks; adducts and ligands are shown in ball-and-stick form with atomic coloring: gray, C; red, O; blue, N; orange, P. Hydrogen bonds are shown as dashed lines.

Figure 7.
Fig. 7. Proposed reactions at the synthetase active site. (a) Superposition of synthetase active sites from crystal structures with informative ligands. The A. aeolicus GatCAB with ATP, Mn^2+, and Asp (this work) is shown in green with yellow C atoms for the ligands; the S. aureus GatCAB with ADP and Mg^2+^14 in orange with orange C for the ligands; and the M. thermautotrophicus GatDE with tRNA^15 in magenta with yellow C for tRNA. Mn^2+ ions are shown as purple spheres, Mg^2+ in orange, and water molecules as red spheres. Substrates ATP, ADP, Asp, and 3′-CCA of tRNA^Gln are represented as sticks. Residues interacting with substrates are represented by thick lines. The ammonia channel (gray surface) enters the synthetase active site from the right and is continuous with the tRNA binding site. (b) Model for the activation reaction. ATP is positioned as in the structure reported here. The terminus of Asp-tRNA^Asn was modeled based on the GatDE–tRNA complex in which the 3′-terminal A was disordered. The Asp carboxyl group is coordinated by the metal in the permanent site, as in the Asp complex. (c) Model for the amidation complex. The activated substrate, phosphoryl-Asp-tRNA^Asn, is shifted so that both phosphate and O^δ coordinate the permanent metal, thereby positioning the Asp C^γ atom at the exit of the ammonia tunnel, ready to receive ammonia from the amidase active site. (d) Schematic diagram of the reaction steps depicted in (b) and (c).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 391, 703-716) copyright 2009.