A Novel Dimeric Structure of the RimL Nα-acetyltransferase from Salmonella typhimurium

Matthew W Vetting,Luiz Pedro S De Carvalho,Steven L Roderick,John S Blanchard

doi:10.1074/jbc.m502401200

Abstract

RimL is responsible for converting the prokaryotic ribosomal protein from L12 to L7 by acetylation of its N-terminal amino group. We demonstrate that purified RimL is capable of posttranslationally acetylating L12, exhibiting a V(max) of 21 min(-1). We have also determined the apostructure of RimL from Salmonella typhimurium and its complex with coenzyme A, revealing a homodimeric oligomer with structural similarity to other Gcn5-related N-acetyltransferase superfamily members. A large central trough located at the dimer interface provides sufficient room to bind both L12 N-terminal helices. Structural and biochemical analysis indicates that RimL proceeds by single-step transfer rather than a covalent-enzyme intermediate. This is the first structure of a Gcn5-related N-acetyltransferase family member with demonstrated activity toward a protein N(alpha)-amino group and is a first step toward understanding the molecular basis for N(alpha)acetylation and its function in cellular regulation.

Highlights

The posttranslational acetylation of N␣-protein termini is a common occurrence in eukaryotic proteins where between 50 and 90% of proteins are N␣-acetylated [1]
Cotranslational N␣-acetylation appears to be critical to the function and stability of numerous eukaryotic proteins [15], no known function has been ascribed to the acetylation of prokaryotic ribosomal proteins
Cloning and Expression of rimLWT, rimLC134A, and L12—The rimL gene was amplified by PCR using genomic DNA from S. typhimurium strain MM1278 and cloned into pET-28a(ϩ) (Novagen), which produced RimL fused to an N-terminal thrombin-cleavable hexahistidine tag

Summary

Introduction

The posttranslational acetylation of N␣-protein termini is a common occurrence in eukaryotic proteins where between 50 and 90% of proteins are N␣-acetylated [1]. Incubation of either the tagged or thrombin-cleaved forms with Ac-CoA resulted in additional higher masses (23,444.0 and 21,654.4 Da, respectively), representing 10 –15% protein sample.

Results

Conclusion