Expression in Escherichia coli of N- and C-terminally deleted human holocarboxylase synthetase. Influence of the N-terminus on biotinylation and identification of a minimum functional protein.
Biotin functions as a covalently bound cofactor of biotindependent carboxylases. Biotin attachment is catalyzed by biotin protein ligases, called holocarboxylase synthetase in mammals and BirA in prokaryotes. These enzymes show a high degree of sequence similarity in their biotinylation domains but differ markedly in the length and sequence of their N terminus. BirA is also the repressor of the biotin operon, and its DNA attachment site is located in its N terminus. The function of the eukaryotic N terminus is unknown. Holocarboxylase synthetase with N- and C-terminal deletions were evaluated for the ability to catalyze biotinylation after expression in Escherichia coli using bacterial and human acceptor substrates. We showed that the minimum functional protein is comprised of the last 349 of the 726-residue protein, which includes the biotinylation domain. Significantly, enzyme containing intermediate length, N-terminal deletions interfered with biotin transfer and interaction with different peptide acceptor substrates. We propose that the N terminus of holocarboxylase synthetase contributes to biotinylation through N- and C-terminal interactions and may affect acceptor substrate recognition. Our findings provide a rationale for the biotin responsiveness of patients with point mutations in the N-terminal sequence of holocarboxylase synthetase. Such mutant enzyme may respond to biotin-mediated stabilization of the substrate-bound complex.
Campeau
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Department of Biology, , McGill University Health Center-Montreal Children's Hospital Research Institute, Montréal, Québec H3Z 2Z3, Canada.
Gravel
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RA
D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans
D017931:DNA Primers; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases