Faculty of Advanced Life Sciences, Hokkaido University, Japan
Akiyoshi Nakamura, Min Yao, Sarin Chimnaronk, Naoki Sakai, ○Isao Tanaka
The formation of Gln-tRNAGln differs among the three domains of life. Most bacteria employ
an indirect pathway to produce Gln-tRNAGln by an amidotransferase (Glu-AdT) that acts on
the mis-acylated Glu-tRNAGln. Bacterial Glu-AdTs are heterotrimeric proteins composed of A, B,
and C subunits, and are named GatCAB. GatCAB converts Glu-tRNAGln into Gln-tRNAGln by initially
activating Glu-tRNAGln into γ -phosphoryl-Glu-tRNAGln at the expense of ATP, which is subsequently
transamidated into Gln-tRNAGln using ammonia generated by hydrolysis of glutamine.
The glutaminase and transamidase reactions are tightly coupled and intrinsically dependent on
the binding of Glu-tRNAGln to GatCAB.
In this study, we describe the crystal structures of intact GatCAB complex from Staphylococcus aureus, in the apo form, and in the complexes with glutamine, asparagine, Mn2+ and adenosine triphosphate analog. Based on the structure of GatCAB/glutamine complex, we demonstrated that the glutaminase reaction occurs at the Ser-cis-Ser-Lys catalytic scissors of GatA without the conformational rearrangement of the glutaminase active site. The structure of GatCAB/ADP complex revealed the precise position and environment of the active site at the bottom of the cradle domain of GatB. Two identified catalytic centers for the glutaminase and transamidase reactions are markedly distant but connected by a hydrophilic ammonia channel 30 A in length. Further, we showed the identity elements essential for discrimination of tRNAGln, and proposed a complete model for the overall concerted reactions to synthesize Gln-tRNAGln.