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 a ll organisms. Their biotinylation is catalyzed by holoenzyme synthetas es, which attach a biotin molecule to a specific lysine residue on the apoenzymes. The sequence flanking the biotin binding site is highly c onserved among biotin-dependent enzymes. This sequence conservation mi ght be related to the extensive cross-species activity showed by holoe nzyme 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 dete rmine the sequence requirements for biotinylation by the bacterial hol oenzyme synthetase. We show that the carboxyl-terminal 67 amino acids of PCC-alpha act as an independent domain in the biotinylation reactio n. Mutations that affect several conserved Gly residues and a Pro-Met- Pro sequence near the biotin binding site are critical for biotinylati on. Substitution of the amino acids that flank the biotin acceptor Lys residue or elimination of the last 3 amino acids of the PCC-alpha pep tides had little or no effect on their biotinylation despite their hig h conservation in biotin enzymes.