The Helicobacter pylori Amidotransferase GatCAB Is Equally Efficient in Glutamine-dependent Transamidation of Asp-tRNAAsn and Glu-tRNAGln

Abstract

The amide aminoacyl-tRNAs, Gln-tRNA Gln and Asn-tRNA Asn , are formed in many bacteria by a pretranslational tRNAdependent amidation of the mischarged tRNA species, Glu-tRNA Gln or Asp-tRNA Asn . This conversion is catalyzed by a heterotrimeric amidotransferase GatCAB in the presence of ATP and an amide donor (Gln or Asn). Helicobacter pylori has a single GatCAB enzyme required in vivo for both Gln-tRNA Gln and Asn-tRNA Asn synthesis. In vitro characterization reveals that the enzyme transamidates Asp-tRNA Asn and Glu-tRNA Gln with similar efficiency (k cat /K m of 1368.4 s ؊1 /mM and 3059.3 s ؊1 /mM respectively). The essential glutaminase activity of the enzyme is a property of the A-subunit, which displays the characteristic amidase signature sequence. Mutations of the GatA catalytic triad residues (Lys 52 , Ser 128 , Ser 152 ) abolished glutaminase activity and consequently the amidotransferase activity with glutamine as the amide donor. However, the latter activity was rescued when the mutant enzymes were presented with ammonium chloride. The presence of Asp-tRNA Asn and ATP enhances the glutaminase activity about 22-fold. H. pylori GatCAB uses the amide donor glutamine 129-fold more efficiently than asparagine, suggesting that GatCAB is a glutamine-dependent amidotransferase much like the unrelated asparagine synthetase B. Genomic analysis suggests that most bacteria synthesize asparagine in a glutamine-dependent manner, either by a tRNA-dependent or in a tRNA-independent route. However, all known bacteria that contain asparagine synthetase A form Asn-tRNA Asn by direct acylation catalyzed by asparaginyl-tRNA synthetase. Therefore, bacterial amide aminoacyl-tRNA formation is intimately tied to amide amino acid metabolism.

Natural insertion of the bro-1 β-lactamase gene into the gatCAB operon affects Moraxella catarrhalis aspartyl-tRNAAsn amidotransferase activity

“…In this study, the gene corresponding to M. catarrhalis tRNA Asn was artificially formed by hybridizing two phosphorylated oligonucleotides, 59-AATTCGGGGTATCGCCAAGTTGGTAAGGCATCAGGTTTTGAT-CCTGACATTCGTTGGTTCGAGTCCAGCTACCTCTGCCACTGC-A-39 and 59-GTGGCAGAGGTAGCTGGACTCGAACCAACGAAT-GTCAGGATCAAAACCTGATGCCTTACCAACTTGGCGATACCC-CG-39, and the gene was inserted into the sites EcoRI/PstI of pGFIB to create the pGFIB/MctRNA Asn clone. M. catarrhalis tRNA Asn was prepared according to Sheppard et al (2007). Analysis by electrophoresis on a 12 % polyacrylamide gel containing 8 M urea revealed that more than 99 % of the material was tRNA; its acceptor activity for Asn varied from 11 to 25 %.…”

“…Similarly, most bacteria and all archaea lack GlnRS and use an ND-GluRS and GatCAB or GatDE as a Glu-tRNA AdT (Glu-AdT) to form Gln-tRNA Gln (Curnow et al, 1997;Horiuchi et al, 2001;Sheppard et al, 2007Sheppard et al, , 2008; reviewed by Huot et al, 2010).…”

“…Bacterial GatCABs catalyse three distinct reactions during the transamidation process: they activate Asp-tRNA Asn by phosphorylating the beta-carboxyl group of the aspartyl residue linked to tRNA Asn ; they use NH 4 + directly or hydrolyse the amide group of Gln or Asn to release ammonia, and finally they amidate the activated AsptRNA Asn intermediate into Asn-tRNA Asn (Decicco et al, 2001;Feng et al, 2005;Horiuchi et al, 2001;Sheppard et al, 2007). The GatA subunit belongs to the amidase family and uses Gln or Asn as amide donor (Nakamura et al, 2006;Strauch et al, 1988;Wu et al, 2009).…”

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

“…3 GatCAB is present in most bacteria and archaea. 2,3,6 In vitro, the bacterial GatCAB can transamidate both Glu-tRNA Gln and Asp-tRNA Asn . 2, 12-15 Its role in vivo is determined by which mischarged tRNA species (Glu-tRNA Gln and/or Asp-tRNA Asn ) is present.…”

“…[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.…”