Structure-activity relationships of methionyl-tRNA synthetase: graphics modelling and genetic engineering

Abstract

The 3D structure of the methionyl-tRNA synthetase from E. coli has been investigated using X-ray analysis 1,2 at a resolution 1.8Å. 90% of the molecule is now well defined and the zinc atom has been identified in a buried region of the molecule, close to the active site. At the same time, the refinement of the complex ATP-MetRS at 2.5 Å has been carried out. The crystallographic R factor has been assigned a value of 25% at 2.5Å with an overall temperature factor of 9Å 2 and 22% when the individual temperature factors are refined. A Fourier difference map clearly reveals the electron density of the bound ATP, showing the phosphate groups deeply plunging into the active site. In parallel, the synthetase gene has been used to probe some of the enzyme structure-activity relationships. A series of 60 modified enzymes truncated at the C-terminus have been constructed in vitro and assayed for activity. In agreement with the graphics model, the results show that a minimum of 534 residues is necessary to sustain the aminoacylation reaction. A programme of site-directed mutagenesis is in progress: residues thought to be important for the catalytic activity, the metal coordination and tRNA interaction are being modified. Preliminary results are discussed in the light of the crystallographic model.