The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.
We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.
Li
S J
SJ
Department of Microbiology, University of Illinois, Urbana 61801.
Cronan
J E
JE
D000103:Acetyl-CoA Carboxylase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D015152:Blotting, Northern; D019731:Carbon-Nitrogen Ligases; D002352:Carrier Proteins / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008025:Ligases / Q000235:genetics*; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics; D015183:Restriction Mapping; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D002352:Carrier Proteins; D004269:DNA, Bacterial; C017071:biotin-binding proteins; D012313:RNA; D001710:Biotin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019791:biotin carboxylase; D000103:Acetyl-CoA Carboxylase