SUBSTRATE RECOGNITION BY THE LEUCYL PHENYLALANYL-TRANSFER-RNA-PROTEINTRANSFERASE - CONSERVATION WITHIN THE ENZYME FAMILY AND LOCALIZATION TO THE TRYPSIN-RESISTANT DOMAIN/

The leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) fro m Escherichia coli catalyzes a peptidyltransferase reaction that resul ts in the N-terminal aminoacylation of acceptor proteins using Leu-, P he-, and Met-tRNAs as amino acid donors, We demonstrated that L/F-tran sferase homologs are widely distributed throughout the eubacteria, sup porting our proposal that the enzyme family is ancient and catalyzes e arly peptide bond synthesis, However, here we present data suggesting that the L/F-transferase is not a homolog of the peptidyltransferase e nzymes involved in cell wall peptidoglycan biosynthesis in Gram-positi ve species, such as Staphylococcus aureus. A sequence comparison of th e known L/F-transferase homologs began to identify the essential resid ues required to catalyze a peptidyltransferase reaction and revealed t hat <20% of the residues were invariant within the L/F-transferase fam ily, Despite this sequence variation, substrate specificity was broadl y conserved, and L/F-transferase homologs from Providencia stuartii, V ibrio cholerae, Neisseria gonorrhoeae, and the cyanobacterium Synechoc ystis sp, all complemented an E. coli aat mutant (lacking L/F-transfer ase activity) for the degradation of N-end rule substrates, In vitro c omparison of the most divergent L/F-transferase homologs, from E. coli and the cyanobacterium Synechocystis sp., revealed near-complete cons ervation of both substrate specificity and secondary structure. Finall y, we demonstrated that variants of the E. coli L/F-transferase, lacki ng either 33 or 78 N-terminal residues, retained measurable peptidyl-t ransferase activity and wild type substrate specificity, Overall, our results identified an essential core of an L/F-transferase and reveale d that a peptidyltransferase catalyst may be constructed from similar to 120 amino acids.