Mutagenesis affecting the carboxyl terminus of the biotinyl subunit of transcarboxylase. Effects on biotination.
Biotin is added to biotin-containing enzymes as a post-translational modification catalyzed by holoenzyme synthetase. This reaction is fairly general in that synthetase from one organism will modify enzymes from heterologous sources. This suggests that the polypeptides share some structural characteristic(s) that define(s) them as biotin enzymes. We have reported previously that when the gene coding for the 1.3 S biotinyl subunit of transcarboxylase is expressed in Escherichia coli, the polypeptide produced is biotinated by the cellular synthetase. Using in vitro mutagenesis of this gene, we have begun to define the primary structure involved in the enzymatic addition of biotin to a lysine residue. We show here that the carboxyl terminus of the 1.3 S subunit is critical in biotination. Mutations affecting the COOH-terminal residue do not influence the modification, but elimination of the hydrophobic side chain of the penultimate residue abolishes biotin addition.
D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases; D019878:Carboxyl and Carbamoyl Transferases; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D008025:Ligases / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D001710:Biotin; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; C038308:GGWCC-specific type II deoxyribonucleases; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases