Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria

Abstract

Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X‐100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate‐binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X‐100 and purified by affinity chromatography on monomeric avidin‐Sepharose. The enzyme is composed of three types of polypeptides: the group of α chains (Mr 120000–140000) containing the biotin, the β chain (60000) and an apparently hydrophobic γ chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The β and γ chains but not the α chain could be labelled by N‐ethyl‐[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000–20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl‐CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.

The sodium pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. Specific cleavage by n-alkanols

“…As observed earlier, staining of they chain with Coomassie blue was weak as compared to the p chain [2]. Application of the silver method [13], however, afforded bands with equal intensities (Fig.…”

“…After 5 min at ambient temperature the reaction was completed by further addition of a few milligrams of unlabelled borohydride. The solution, acidified to pH 5 with 1 M acetic acid, contained 4.1 pmol (51 %) (S)-3-hydroxybutyryl-CoA [2] but no acetoacetyl-CoA. Purification was achieved by a passage through the protonated form of Dowex-50 followed by evaporation to dryness and chromatography on DEAE-Sephacel, chloride form (column dimensions 1.5 x 10 cm) (Pharmacia, Sweden).…”

“…The ct chains contain biotin and the y chain may be involved in the Na ' transport since this ion specifically protects the peptide from tryptic digestion [2].…”

“…Recently this biotin-dependent decarboxylase was characterized as a sodium pump [l]. It was solubilized by Triton X-100 at high ionic strength, purified by affinity chromatography on avidinSepharose and reconstituted by incorporation into lipid vesicles [2]. The enzyme from Acidaminococcus fermentans consists of three different polypeptide chains (a, M , 120000; B, M , 60000 and y, M , 30000).…”

“…A mixture containing 90 -95% N-carboxybiotin dimethyl ester (isomer ratio, 1'-N:3'-N = 15:l) and 5-10% biotin methyl ester was synthesized according to [7]. The purification of glutaconyl-CoA decarboxylase and the photometrical assay were described previously [2]. Glutaconate CoA-transferase [8] and crotonase [9] were prepared, recrystallized and assayed by established procedures.…”