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J. Biol. Chem., Vol. 267, Issue 2, 855-863, Jan, 1992
SJ Li and JE Cronan Jr
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.
The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase
Department of Microbiology, University of Illinois, Urbana 61801.
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