Holocarboxylase synthetase (HCS) is an essential enzyme that catalyzes the incorporation of biotin into apo carboxylase and the biotinylation of the four biotin-dependent carboxylases in the human cell Deficiency of HCS results in decreased activity of these carboxylases and affects various metabolic processes Despite the importance of this enzyme, the recognition mechanism of the biotinoyl domain by human HCS (hHCS) has remained unclear We have developed a method to express hHCS in the baculovirus system and used it to purify catalytically active, full-length hHCS NMR experiments oil the biotinoyl domains from acetyl-CoA carboxylase indicate that when hHCS is added, it recognizes the MKM motif in human and in Escherichia coli with a preference to the human biotinoyl domain In addition, hHCS can biotinylate the biotinoyl domains from human and E colt acetyl-CoA carboxylase at similar rates compared to the E. coli biotin protein ligase. Bit-A. which reacts very slowly with the human biotinoyl domain We propose that the hHCS has greater substrate acceptability, while the BirA has higher Substrate specificity These results provide insights into Substrate recognition by hHCS, which can be distinguished from BirA in this respect (C) 2009 Elsevier Inc All rights reserved.