The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase

S. J. Li, J. E. Cronan

Research output: Contribution to journalArticlepeer-review

Abstract

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 α-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 β-galactosidase resulted in biotinated β-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.

Original languageEnglish (US)
Pages (from-to)855-863
Number of pages9
JournalJournal of Biological Chemistry
Volume267
Issue number2
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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