Novel type of ADP-forming acetyl coenzyme A synthetase in hyperthermophilic archaea: heterologous expression and characterization of isoenzymes from the sulfate reducer Archaeoglobus fulgidus and the methanogen Methanococcus jannaschii

Abstract

Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) right harpoon over left harpoon acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers (alpha(2), beta(2)). The coding genes for the alpha and beta subunits are located at different sites in the P. furiosus chromosome. Based on significant sequence similarity of the P. furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the alpha and beta subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus and ORF MJ0590 of M. jannaschii were cloned and functionally overexpressed in Escherichia coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P. furiosus alpha and beta subunits in an alphabeta (AF1211 and MJ0590) or betaalpha (AF1938) orientation. The data indicate that A. fulgidus and M. jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused.

FIG. 1.

Comparison of deduced amino acid sequences of the α subunit (462 aa) and the β subunit (232 aa) of ACD I from P. furiosus with ACD II from P. furiosus (457 and 238 aa) (Utah Genome Center Website); ORF AF1211 (685 aa), AF1938 (673 aa), AF1511 (881 aa), AF1192 (664 aa), and AF0932 (666 aa) from A. fulgidus strain VC16 (8); ORF MJ0590 (704 aa) from M. jannaschii (5); ORF YFIQ from E. coli (886 aa) (14); ORF AF107206 from G. lamblia (726 aa) (7); and ORF AF286346 from Entamoeba histolytica (713 aa) (6) (deduced from genome sequences). The α and β subunits of P. furiosus ACD I and the homologous domains of the putative proteins are shown in white and shaded boxes, respectively. The amino acid sequence identities of the domains to the α and β subunits of P. furiosus ACD I are shown in white and shaded boxes, respectively. Thin lines represent areas without recognized homologies.

FIG. 2.

Purified recombinant A. fulgidus ACD isoenzymes, isolated from transformed E. coli, as analyzed by SDS-PAGE. The protein was separated on a 12% polyacrylamide gel and subsequently stained with Coomassie brilliant blue R250 (10). Lanes: 1, molecular mass standard (Sigma); 2, 0.9 μg of purified recombinant ACD-AF1211; 3, 0.9 μg of purified recombinant ACD-AF1938.

FIG. 3.

Rate dependence of purified recombinant A. fulgidus ACD-AF1211 activity on the acetyl-CoA concentration at 55°C. The inset shows a double-reciprocal plot of the rate against the corresponding substrate concentration. The assay mixture contained 0.36 μg of enzyme.

FIG. 4.

Effect of temperature on the specific activity of purified recombinant A. fulgidus ACD-AF1211. (A) Temperature dependence of the specific activity; (B) Arrhenius plot of the same data. Enzyme activity was measured in the direction of acetate formation (see Materials and Methods). The assay mixture contained 0.36 μg of enzyme.

FIG. 5.

Thermostability of purified recombinant A. fulgidus ACD-AF1211. A 0.3-μg portion of enzyme was incubated in 60 μl of EPPS (pH 8.0) at 70°C (▪), 80°C (•), or 85°C (▴). At the times indicated, 25-μl aliquots were withdrawn and assayed for remaining activity at 55°C in the direction of acetate formation. 100% activity corresponded to an ACD-AF1211 specific activity of 60 U/mg.

FIG. 6.

Rate dependence of purified recombinant A. fulgidus ACD-AF1938 activity on the phenylacetyl-CoA concentration at 55°C. The inset shows a double-reciprocal plot of the rate against the corresponding substrate concentration. The assay mixture contained 0.36 μg of enzyme.

FIG. 7.

RT-PCR from cDNA amplified with specific primers to detect the mRNA formation after transcriptions of the gene AF0623 (DNA ligase) and the ORFs AF1211 (coding for ACD I) and AF1938 (coding for ACD II) from A. fulgidus strain 7324. Lanes: 1, marker (Fermentas); 2, AF0623 (control, DNA ligase); l4, AF1211; l6, AF1938; l3, 5, and 7, PCR from total RNA without transciption in cDNA through the RT (negative controls).

FIG. 8.

Purified recombinant M. jannaschii ACD-MJ0590, isolated from transformed E. coli cells, as analyzed by SDS-PAGE. The protein was separated on a 12% polyacrylamide gel and subsequently stained with Coomassie brilliant blue R250 (10). Lanes: 1, molecular mass standard (Sigma); 2, 1.6 μg of purified recombinant ACD-MJ0590.

FIG. 9.

Proposed physiological role of the A. fulgidus ACD isoenzymes ACD-AF1211 (ACD I) and ACD-AF1938 (ACD II) in the sugar and peptide metabolism, by analogy to the P. furiosus ACD isoenzymes I and II (11). TA, transaminase; POR, pyruvate:ferredoxin oxidoreductase; VOR, 2-ketoisovalerate:ferredoxin oxidoreductase, IOR, indolepyruvate:ferredoxin oxidoreductase.