Abstract
The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphyloccocus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic β-bulge splayed at the β-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.
Highlights
The Gcn5-related N-acetyltransferases (GNATs) are a very large enzyme superfamily with more than 10,000 members identified across all kingdoms of life [1]
A signature of the GNAT fold is a splay between b4 and b5 strands, forming a V-shape opening in the central b sheet which is crucial in the transfer of acetyl group and binding of acetyl-CoA [7]
Protein production and structure determination To determine the x-ray crystallographic structure of SaGNAT, the gene encoding the protein was cloned into bacterial expression vector pMCSG21 [20] and recombinantly expressed as a 6-His tagged fusion protein in E. coli BL21 (DE3) pLysS
Summary
The Gcn5-related N-acetyltransferases (GNATs) are a very large enzyme superfamily with more than 10,000 members identified across all kingdoms of life [1]. Motif D, encompassing b2 and b3 strands is not directly involved in substrate or cofactor binding, but stabilises core structural elements of the protein, while motif C, located at the N-terminus of the protein, is the least conserved with some histone N-acetyltransferases lacking this motif completely. These motifs together comprise the common structural core known as GNAT domain. Our structure confirms that the protein exhibits many of the classical GNAT motifs, has high structural similarity with the phosphinoacetyl GNAT proteins, and is likely to exist as a dimer in solution based on biophysical and crystallographic properties
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