THE STRUCTURAL BASIS FOR SERYL-ADENYLATE AND AP(4)A SYNTHESIS BY SERYL-TRANSFER-RNA SYNTHETASE

Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthet ase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg . ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently tr ansferred to the 3'-end of the tRNA. In common with most other aminoac yl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap(4)A) from the enzyme-bound adenylate in termediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of ami noacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis i n all living cells. Results: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermopilus are described. The firs t complex is of the enzyme with ATP and Mn2+. The ATP is found in an u nusual bent conformation, stabilized by interactions with conserved ar ginines and three manganese ions. The second complex contains seryl-ad enylate in the active site, enzymatically produced in the crystal afte r soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap(4)A and Mn2+ All three structures exhibit a common Mn2+ sit e in which the cation is coordinated by two active-Site residues in ad dition to the alpha-phosphate group from the bound ligands. Conclusion s: Superposition of these structures allows a common;reaction mechanis m for seryl-adenylate and Ap(4)A formation to be proposed. The bent co nformation of the ATP and the position of the serine are consistent wi th nucleophilic attack of the serine carboxyl group on the alpha-phosp hate by an in-line displacement mechanism leading to the release of th e inorganic pyrophosphate. A second ATP molecule can bind with its gam ma-phosphate soup in the same position as the beta-phosphate of the or iginal ATP. This can attack the seryl-adenylate with the formation of Ap(4)A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly i nvolved in stabilizing the transition state.