Sequence requirements for the biotinylation of carboxyl-terminal fragments of human propionyl-CoA carboxylase alpha subunit expressed in Escherichia coli.
Biotin-dependent enzymes play an essential role in the metabolism of all organisms. Their biotinylation is catalyzed by holoenzyme synthetases, which attach a biotin molecule to a specific lysine residue on the apoenzymes. The sequence flanking the biotin binding site is highly conserved among biotin-dependent enzymes. This sequence conservation might be related to the extensive cross-species activity showed by holoenzyme synthetases. In this study, we have expressed carboxyl-terminal fragments of the alpha subunit of human propionyl-CoA carboxylase (PCC-alpha) in Escherichia coli and used site-directed mutagenesis to determine the sequence requirements for biotinylation by the bacterial holoenzyme synthetase. We show that the carboxyl-terminal 67 amino acids of PCC-alpha act as an independent domain in the biotinylation reaction. Mutations that affect several conserved Gly residues and a Pro-Met-Pro sequence near the biotin binding site are critical for biotinylation. Substitution of the amino acids that flank the biotin acceptor Lys residue or elimination of the last 3 amino acids of the PCC-alpha peptides had little or no effect on their biotinylation despite their high conservation in biotin enzymes.
Leon-Del-Rio
A
A
McGill University-Montreal Children's Hospital Research Institute, Quebec, Canada.
Gravel
R A
RA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001710:Biotin / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D043586:Methylmalonyl-CoA Decarboxylase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001710:Biotin; D002262:Carboxy-Lyases; D043586:Methylmalonyl-CoA Decarboxylase