Identification of missing genes and enzymes for autotrophic carbon fixation in crenarchaeota

Abstract

Two autotrophic carbon fixation cycles have been identified in Crenarchaeota. The dicarboxylate/4-hydroxybutyrate cycle functions in anaerobic or microaerobic autotrophic members of the Thermoproteales and Desulfurococcales. The 3-hydroxypropionate/4-hydroxybutyrate cycle occurs in aerobic autotrophic Sulfolobales; a similar cycle may operate in autotrophic aerobic marine Crenarchaeota. Both cycles form succinyl-coenzyme A (CoA) from acetyl-CoA and two molecules of inorganic carbon, but they use different means. Both cycles have in common the (re)generation of acetyl-CoA from succinyl-CoA via identical intermediates. Here, we identified several missing enzymes/genes involved in the seven-step conversion of succinyl-CoA to two molecules of acetyl-CoA in Thermoproteus neutrophilus (Thermoproteales), Ignicoccus hospitalis (Desulfurococcales), and Metallosphaera sedula (Sulfolobales). The identified enzymes/genes include succinyl-CoA reductase, succinic semialdehyde reductase, 4-hydroxybutyrate-CoA ligase, bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase, and beta-ketothiolase. 4-Hydroxybutyryl-CoA dehydratase, which catalyzes a mechanistically intriguing elimination of water, is well conserved and rightly can be considered the key enzyme of these two cycles. In contrast, several of the other enzymes evolved from quite different sources, making functional predictions based solely on genome interpretation difficult, if not questionable.

FIG. 1.

Pathways of autotrophic CO₂ fixation in Crenarchaeota. (A) 3-Hydroxypropionate/4-hydroxybutyrate cycle, as proposed for Sulfolobales (see reference and literature cited therein). (B) Dicarboxylate/4-hydroxybutyrate cycle, as proposed for Desulfurococcales and Thermoproteales (24, 41). The enzymes studied here in Metallosphaera sedula, Thermoproteus neutrophilus, and Ignicoccus hospitalis are numbered 10 to 16 below; the identified genes are given in parentheses. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH or reduced methyl viologen) (Msed_0709, Tneu_0421, Igni_?); 11, succinic semialdehyde reductase (NADPH) (Msed_1424, Tneu_0419, Igni_?); 12, 4-hydroxybutyrate-CoA ligase (AMP forming) (Msed_?, Tneu_0420, Igni _?); 13, 4-hydroxybutyryl-CoA dehydratase (Msed_1321, Tneu_0422, Igni_0595); 14, crotonyl-CoA hydratase (Msed_0399, Tneu_0541, Igni _1058); 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺) (Msed_0399, Tneu_0541, Igni _1058); 16, acetoacetyl-CoA beta-ketothiolase (Msed_0656, Tneu_0249, Igni_1401); 17, pyruvate synthase (reduced methyl viologen); 18, pyruvate:water dikinase; 19, PEP carboxylase; 20, malate dehydrogenase (NADH); 21, fumarate hydratase; 22, fumarate reductase (reduced methyl viologen); 23, succinyl-CoA synthetase (ADP forming). Fd_red, reduced ferredoxin; MV, methyl viologen.

FIG. 2.

Transcriptional organization of a gene cluster in T. neutrophilus that is involved in the dicarboxylate/4-hydroxybutyrate cycle. cbs, three genes coding for proteins containing cystathionine β-synthase domains; pepc, PEP carboxylase gene; ssr, succinic semialdehyde reductase gene; 4hbl, 4-hydroxybutyrate-CoA ligase gene; scr, succinyl-CoA reductase gene; 4hbd, 4-hydroxybutyryl-CoA dehydratase gene; frd, fumarate reductase subunits A and B genes. Bar, 500-bp scale.