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

S J Li,J E Cronan

doi:10.1016/s0021-9258(18)48362-7

S J Li, J E Cronan

Open Access

https://doi.org/10.1016/s0021-9258(18)48362-7

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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 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.

Highlights

We report the molecular cloning and DNA sequence carboxylase subunits, biotin carboxylase and carboxyltransof the gene encoding the biotin carboxylase subunit of ferase(Albertsand Vagelos, 1972; Polakis et al, 1974).A
In E. coli acetyl-coA carboxylase and Propionibacterium shermnii transcarboxylase (Samols et al, 1988), the covalently bound biotin, bioticnarboxylase, and carboxyltransferase reside in distinct protein subunits, whereas the higher eucaryotic acetyl-coAcarboxylases (Takai etal., 1988; LopezCasillas et al, 1988) and yeast pyruvate carboxylase (Lim et al, 1988) contain all three components in asingle protein chain
Since most recent mechanistic indicating thatBCCP is a protein of atypical physical studies of biotin carboxylation have used E. coli biotin carproperties

Summary

RESULTS

Galactosidase molecules and production of onesuch Cloning of the Genes Encoding BCCP and Biotin Carboxylfusion was shown to result in derepression of the biotin ase-During previous work on protein biotination This DNA segment was used as a hybridization probe in Southern blots of E. coli. Acetyl-coA carboxylase catalyses the first committed step chromosomal DNA digested with a variety of restriction enin fattyacid synthesis, the synthesiosf malonyl-CoA The overall reactionconsists of two fragments underhighly stringent conditions indicatinga high distinct half-reactions(Scheme l), the carboxylatioonf biotin level of specific base pairing (>85%) between the synthetic with bicarbonate followed by transfer of the CO, group from DNA and a single chromosomalfragment of each digest (data carboxy-biotin to acetyl-coA to form malonyl-CoA (Alberts not shown). 1, 3, and 5) are presented in miniprint at the end of this paper.

ACETYL CoA

DISCUSSION

Findings

Gene Ethnecoding Biotin Carboxylase Subunit