Malonyl-Coenzyme A Reductase in the Modified 3-Hydroxypropionate Cycle for Autotrophic Carbon Fixation in Archaeal Metallosphaera and Sulfolobus spp

Abstract

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO 2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO 2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonylCoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA ؉ NADPH ؉ H ؉ 3 malonate-semialdehyde ؉ CoA ؉ NADP ؉ . In its native state it is associated with small RNA. Its activity was stimulated by Mg 2؉and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (K m ‫؍‬ 25 M) and malonyl-CoA (K m ‫؍‬ 40 M). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonylCoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.

3-Hydroxypropionyl-Coenzyme A Synthetase from Metallosphaera sedula , an Enzyme Involved in Autotrophic CO 2 Fixation

“…In C. symbiosum 3-hydroxypropionate may be activated to its CoA ester by means of an ADP-forming enzyme. The 3-hydroxypropioyl-CoA synthetase described here is the third enzyme of the modified 3-hydroxypropionate cycle for which the corresponding genes have been identified in M. sedula, the other two being acetyl-CoA/propionyl-CoA carboxylase and malonyl-CoA reductase (2,15). Genes encoding the subunits of a biotin-dependent acyl-CoA carboxylase are present in the genome of C. symbiosum and are likely to encode acetyl-CoA/propionyl-CoA carboxylase.…”

“…The cells were harvested by centrifugation at an optical density at 578 nm of 0.2 to 0.3 and stored in liquid nitrogen until used. Sulfolobus tokodaii (DSMZ 16993) was grown aerobically under heterotrophic conditions, and chromosomal DNA was isolated as described previously (2).…”

“…The primary carboxylation steps are catalyzed by a bifunctional acetyl-CoA/propionyl-CoA carboxylase (7,15). Malonyl-CoA, the carboxylation product of acetyl-CoA, is reduced to malonate semialdehyde by malonyl-CoA reductase (2) and is reduced further to the characteristic intermediate of the pathway, 3-hydroxypropionate (21). For C. aurantiacus, a single enzyme catalyzes both reduction steps (16; Fig.…”

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

“…1B). The three enzymes are referred to here as E1 (acetyl/propionyl-CoA carboxylase, encoded by Msed_0147, Msed_0148, and Msed_1375), E2 (malonyl/succinylCoA reductase, Msed_0709), and E3 (malonate semialdehyde reductase, Msed_1993) (18)(19)(20). E1 carboxylates acetyl-CoA using bicarbonate and requires ATP.…”

A dicarboxylate/4-hydroxybutyrate autotrophic carbon assimilation cycle in the hyperthermophilic Archaeum Ignicoccus hospitalis

“…(i) Green nonsulfur bacteria (Chloroflexus aurantiacus and related bacteria) use acetyl-CoA/propionyl-CoA carboxylase(s) as carboxylating enzyme(s) to form succinyl-CoA (route 1), from which acetyl-CoA is regenerated via malyl-CoA cleavage (route 4) and glyoxylate is the carbon fixation product (3-hydroxypropionate/ glyoxylate cycle) (Fig. 4) (14)(15)(16).…”