Molecular dissection of arginyltransferases guided by similarity to bacterial peptidoglycan synthases

Abstract

Post-translational protein arginylation is essential for cardiovascular development and angiogenesis in mice and is mediated by arginyl-transfer RNA-protein transferases Ate1-a functionally conserved but poorly understood class of enzymes. Here, we used sequence analysis to detect the evolutionary relationship between the Ate1 family and bacterial FemABX family of aminoacyl-tRNApeptide transferases, and to predict the functionally important residues in arginyltransferases, which were then used to construct a panel of mutants for further molecular dissection of mouse Ate1. Point mutations of the residues in the predicted regions of functional importance resulted in changes in enzymatic activity, including complete inactivation of mouse Ate1; other mutations altered its substrate specificity. Our results provide the first insights into the mechanisms of Ate1-mediated arginyl transfer reaction and substrate recognition, and define a new protein superfamily called Dupli-GNAT to reflect its origin by the duplication of the GNAT acetyltransferase domain.

Arginyltransferase Is an ATP-Independent Self-Regulating Enzyme that Forms Distinct Functional Complexes In Vivo

“…Since it has been previously reported that out of the four mouse ATE1 isoforms, ATE1-1 and ATE1-2 appear to have higher activity and more universal substrate specificity (Rai and Kashina, 2005; Rai et al, 2006), and ATE1-2 is the most ubiquitous ATE1 isoform in different mouse tissues (Rai and Kashina, 2005), we used ATE1-2 for the initial arginylation assay setup and characterization of the arginyl transfer reaction. We found that mixing purified ATE1-2 with BSA and the components described above results in rapid and efficient incorporation of [ 3 H]-Arg into BSA (Fig.…”

“…A discovery that mammalian Ate1 gene generates a subset of highly homologous but distinct isoforms led to controversial reports about these isoforms' activities and substrate specificities (Hu et al, 2006; Rai and Kashina, 2005; Rai et al, 2006), further suggesting that the arginylation reaction in vivo may be more complex than it appears. To add to the mystery, recent identification of a large number of arginylated proteins in vivo (Wong et al, 2007) raised a multitude of possibilities about the arginylation reaction mechanisms and function.…”

The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase

“…A group of aminoacyl-tRNA protein transferases involved in peptidoglycan biosynthesis have a similar structural fold, but are dissimilar in substrate specificity or function, to L/F transferase (Benson et al 2002;Biarrotte-Sorin et al 2004;Rai et al 2006;Dong et al 2007). Factors essential for methicillin resistance (Fem) transferase X from Weissella viridescens (FemX Wv ) transfers L -Ala from L -Ala-tRNA Ala to UDPMurNAc-pentapeptide (Maillard et al 2005).…”

“…Factors essential for methicillin resistance (Fem) transferase X from Weissella viridescens (FemX Wv ) transfers L -Ala from L -Ala-tRNA Ala to UDPMurNAc-pentapeptide (Maillard et al 2005). The C-terminal domain of L/F transferase belongs to the GCN5-related Nacetyltransferase (GNAT) protein superfamily (Rai et al 2006;Dong et al 2007) and is similar to the two domains of FemX Wv (Biarrotte-Sorin et al 2004). The Ala-tRNA Ala specificity for FemX Wv is mainly through steric hindrance of the aminoacyl moiety, where it excludes most amino acids besides glycine (Fonvielle et al 2009 Gly with the anti-determinant C 2 :G 71 base pair) (Villet et al 2007;Fonvielle et al 2009).…”

An Alternative Mechanism for the Catalysis of Peptide Bond Formation by L/F Transferase: Substrate Binding and Orientation

“…1,2 The aminoacyltRNA protein transferase with ribosome peptidyltransferase-like activity belongs to the dupli-GNAT protein superfamily. 3 One family of enzymes in this superfamily is involved in peptidoglycan synthesis in some Gram-positive bacteria. 4 The focus of this investigation is another family of these enzymes that catalyzes the posttranslational addition of an amino acid to the N-terminus of a protein substrate.…”