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.