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

Abstract

1. The primary sodium-ion pump glutaconyl-CoA decarboxylase (GCD) from Acidaminococcus femzentans is composed of four subunits: GCDA, the carboxytransferase (65 m a ) , GCDB, the carboxylyase (36 m a ) , GCDC, the biotin carrier (24 kDa) and GCDD (14 kDa) of unknown function. A genomic library of A. femzentans 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. femzentans 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 preceeding 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 pJFll8HE. 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 DH5a by moderately inducing the tac promoter of pJFll8HE with 25 -100 pM isopropyl-1-thio-P-D-galactoside. Strong induction (1 mM isopropyl-1-thio-P-D-galactoside) led to the formation of inclusion bodies from which GCDA could not be reactivated. The apparent K, = 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 K, = 40mM). Biocytin was found to be a somewhat better carboxy acceptor for the expressed GCDA subunit (apparent K, = 13 mM; V = l.Onkat/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.

Energetics and biochemistry of fermentative benzoate degradation by Syntrophus gentianae

“…(formerly reported as Pseudomonas strains K172 and KB740; Härtel et al 1993), which catalyze the decarboxylation simultaneously with the glutaryl-CoA dehydrogenase reaction in the cytoplasm without energy conservation, S. gentianae employs a membrane-bound enzyme that is strictly sodium-dependent and likely to couple the decarboxylation with a translocation of sodium ions across the membrane. Such enzymes have been studied in detail with Acidaminacoccus fermentans (Buckel and Semmler 1983;Buckel 1986;Bendrat and Buckel 1993), and a similar enzyme system even acts as the sole energy source for a bacterium running its entire energy metabolism on the basis of glutarate decarboxylation to butyrate and isobutyrate (Matthies and Schink 1992). The sodium ions transported across the cytoplasmic membrane may give rise to ATP formation either directly by a membrane-bound sodium-ion-pumping ATPase (Dimroth 1997) or by a proton-pumping ATPase after translation into a proton gradient through a proton/Na + antiporter.…”

Unusual DAhydrations in anaerobic bacteria: considering ketyls (radical anions) as reactive intermediates in enzymatic reactions

“…The genes from A. fermentans coding for the three polypeptides (a,[3 and 7) are clustered together in All rights reserved. the order hgdCAB, which form the 3'-end of the 'hydroxyglutarate operon', comprising in addition gctA and B coding for glutaconate CoA-tranferase as well as gcdA coding for the hydrophilic carboxytransferase subunit of the biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase, the consecutive enzyme of this pathway [7,17]. The extremely oxygen-sensitive activator HgdC (half-life under air at 25°C approx.…”

A mutation in the indole-3-acetic acid biosynthesis pathway of Pseudomonas syringae pv. syringae affects growth in Phaseolus vulgaris and syringomycin production

“…It is possible that ORF3 encodes a product that is involved in further metabolism of IAA, perhaps functioning to decarboxylate IAA. An alternative explanation is that ORF3 encodes a decarboxylase that is part of an uncoupled ion pump which could be involved in IAA transport (8).…”