title: Rational design of aminoacyl-tRNA synthetase specific for p-acetyl-L-phenylalanine.
abstract: The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations.
authors: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China. renhuachina@msn.com
authors: Renhua
authors: R
authors: Sun
authors: Heng
authors: H
authors: Zheng
authors: Zhengzhi
authors: Z
authors: Fang
authors: Wenbing
authors: W
authors: Yao
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D017017:Methanococcus / Q000201:enzymology*; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D015202:Protein Engineering; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C476995:3-(4-acetylphenyl)-2-aminopropanoic acid; D014443:Tyrosine; D010649:Phenylalanine; D014448:Tyrosine-tRNA Ligase