Mycobacterium smegmatis is a good model for studying the physiology and pathogenesis of Mycobacterium tuberculosis due to its genetic similarity. As methionine biosynthesis exists only in microorganisms, the enzymes involved in methionine biosynthesis can be a potential target for novel antibiotics. Homoserine O-acetyltransferase from M. smegmatis (MsHAT) catalyzes the transfer of acetyl-group from acetyl-CoA to homoserine. To investigate the molecular mechanism of MsHAT, we determined its crystal structure in apo-form and in complex with either CoA or homoserine and revealed the substrate binding mode of MsHAT. A structural comparison of MsHAT with other HATs suggests that the conformation of the alpha 5 to alpha 6 region might influence the shape of the dimer. In addition, the active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HATs. (C) 2019 Elsevier Inc. All rights reserved.