Structural Basis for tRNA-Dependent Amidotransferase Function

Abstract

Besides direct charging of tRNAs by aminoacyl-tRNA synthetases, indirect routes also ensure attachment of some amino acids onto tRNA. Such routes may explain how new amino acids entered into protein synthesis. In archaea and in most bacteria, tRNA(Gln) is first misaminoacylated by glutamyl-tRNA synthetase. Glu-tRNA(Gln) is then matured into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report the structure of a tRNA-dependent amidotransferase-that of GatDE from Pyrococcus abyssi. The 3.0 A resolution crystal structure shows a tetramer with two GatD molecules as the core and two GatE molecules at the periphery. The fold of GatE cannot be related to that of any tRNA binding enzyme. The ammonium donor site on GatD and the tRNA site on GatE are markedly distant. Comparison of GatD and L-asparaginase structures shows how the motion of a beta hairpin region containing a crucial catalytic threonine may control the overall reaction cycle of GatDE.

On the Evolution of the tRNA-Dependent Amidotransferases, GatCAB and GatDE

“…1). 3,[21][22][23]31 The latter enzymes are encoded in the nuclear genomes of many eukaryotes. 31 In Saccharomyces cerevisiae, Pet112 is essential for mitochondrial translation.…”

“…It shows homology to the tail domain of both subunits. [21][22][23] YqeY is found in a diverse array of bacteria across multiple phyla but their function remains unknown. 42 A YqeY domain appended to the C-terminus of the Deinococcus radiodurans GlnRS is important for the enzyme to productively bind to tRNA Gln .…”

“…[20][21][22] 2) The AdTs have a glutaminase subunit (GatA and GatD, respectively) to liberate ammonia from an amide donor such as Gln or Asn. 2,20,23,24 GatD belongs to the type I L-asparaginase (AnsA) family 3, 20, 23 while GatA is an amidase. 2, 10, 21, 24 GatC, a small protein of approximately 100 amino acids, is required by GatA for proper folding 10 and binding to GatB.…”

Plasmodium Apicoplast Gln-tRNAGln Biosynthesis Utilizes a Unique GatAB Amidotransferase Essential for Erythrocytic Stage Parasites

“…The spatial arrangement of PfGatA and PfGatB was determined based on the crystal structures of S. aureus Gat-CAB. Attempts to model GatA and GatB according to the crystal structure of Pyrococcus abyssi archaeal GatDE (1zq1) (31) resulted in unusable models. All figures were prepared with Chimera (32).…”

“…1) includes an N-terminal apicoplast targeting sequence (residues 1-183), two unique inserts (residues 184 -362 and 461-524) of unknown functions, and two domains typical for bacterial GatB, the cradle domain (residues 350 -460 and 526 -700), and a helical domain (residues 704 -880). Instead of GatB, archaea use GatE, which contains an AspRS-like insertion in its cradle domain (31). No similarity could be found between the Plasmodium and archaeal insertions.…”