Substrate recognition characteristics of human holocarboxylase synthetase for biotin ligation.
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 on 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. coli acetyl-CoA carboxylase at similar rates compared to the E. coli biotin protein ligase, BirA, 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.
Lee
Chung-Kyung
CK
Division of Magnetic Resonance Research, Korea Basic Science Institute, Cheongwon-Gun, Chungbuk 363-883, Republic of Korea.
Cheong
Chaejoon
C
Jeon
Young Ho
YH
D000103:Acetyl-CoA Carboxylase; D000595:Amino Acid Sequence; D001360:Avidin / Q000378:metabolism; D016367:Baculoviridae; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D001360:Avidin; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases; C058125:birA protein, E coli; D000103:Acetyl-CoA Carboxylase