Cloning, sequencing and expression of the gene encoding the carboxytransferase subunit of the biotin-dependent Na+ pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans in Escherichia coli.
1. The primary sodium-ion pump glutaconyl-CoA decarboxylase (GCD) from Acidaminococcus fermentans is composed of four subunits: GCDA, the carboxytransferase (65 kDa), GCDB, the carboxylyase (36 kDa), GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. fermentans was screened with an antiserum raised against whole GCD. A clone giving the strongest reaction in an immunoassay contained a 12-kbp genomic fragment from A. fermentans and was analysed further. An oligonucleotide deduced from the N-terminus of GCDA was used for probing the corresponding gene gcdA. It is 1761 bp in length and encodes for a protein of 64.3 kDa. Both partial amino acid sequences obtained from GCDA, the N-terminus as well as an internal tryptic peptide, were detected in the open reading frame (ORF) of gcdA. 2. Sequencing of the flanking regions revealed three adjacent ORF (ORF1-3) which do not code for any of the peptide sequences known of the other GCD subunits. The ORF downstream of gcdA (ORF3) is followed by hgdA and hgdB coding for 2-hydroxyglutaryl-CoA dehydratase, the preceding enzyme of the pathway of glutamate fermentation. Our results suggest that at least these three genes of the hydroxyglutarate pathway are organised in an operon and that the genes of the other GCD subunits from which peptide sequences are known (GCDB and GCDC) are not located adjacent to gcdA. 3. gcdA was amplified from genomic DNA using the polymerase chain reaction and cloned into the expression vector pJF118HE. Active GCDA subunit (up to 2.8 nkat/mg protein), catalysing the biotin-dependent formation of crotonyl-CoA from glutaconyl-CoA, was obtained in cell-free extracts of Escherichia coli DH5 alpha by moderately inducing the tac promoter of pJF118HE with 25-100 microM isopropyl-1-thio-beta-D-galactoside. Strong induction (1 mM isopropyl-1-thio-beta-D-galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent Km = 51 mM for free biotin of the expressed GCDA subunit with V = 1.9 nkat/mg protein is similar to that of butanol-treated GCD composed of GCDA and GCDC (apparent Km = 40 mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent Km = 13 mM; V = 1.0 nkat/mg protein). 4. Native GCD and expressed GCDA were treated with 2 mM N-ethylmaleimide showing different kinetics of inactivation: GCD lost half of its activity within 6 min, whereas expressed GCDA required 21 min.
Bendrat
K
K
Laboratorium für Mikrobiologie, Philipps-Universität Marburg, Federal Republic of Germany.
Buckel
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D001710:Biotin / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005033:Ethylmaleimide / Q000494:pharmacology; D015870:Gene Expression; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics; D014169:Transformation, Bacterial; D045854:Veillonellaceae / Q000201:enzymology / Q000235:genetics*
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D001710:Biotin; D002262:Carboxy-Lyases; C023060:glutaconyl CoA decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase; D005033:Ethylmaleimide