Abstract
Members of the GCN5-related N-acetyltransferase (GNAT) family are found in all domains of life and are involved in processes ranging from protein synthesis and gene expression to detoxification and virulence. Due to the variety of their macromolecular targets, GNATs are a highly diverse family of proteins. Currently, 3D structures of only a small number of GNAT representatives are available and thus the family remains poorly characterized. Here, the crystal structure of the guanidine riboswitch-associated GNAT from Lactobacillus curiae (LcGNAT) that acetylates canavanine, a structural analogue of arginine with antimetabolite properties, is reported. LcGNAT shares the conserved fold of the members of the GNAT superfamily, but does not contain an N-terminal β0 strand and instead contains a C-terminal β7 strand. Its P-loop, which coordinates the pyrophosphate moiety of the acetyl-coenzyme A cosubstrate, is degenerated. These features are shared with its closest homologues in the polyamine acetyltransferase subclass. Site-directed mutagenesis revealed a central role of the conserved residue Tyr142 in catalysis, as well as the semi-conserved Tyr97 and Glu92, suggesting that despite its individual substrate specificity LcGNAT performs the classical reaction mechanism of this family.