Autotrophic Carbon Dioxide Assimilation in Thermoproteales Revisited

Abstract

For Crenarchaea, two new autotrophic carbon fixation cycles were recently described. Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the carboxylating enzyme. Ignicoccus hospitalis (Desulfurococcales) uses the dicarboxylate/4-hydroxybutyrate cycle, with pyruvate synthase and phosphoenolpyruvate carboxylase being responsible for CO 2 fixation. In the two cycles, acetyl-CoA and two inorganic carbons are transformed to succinyl-CoA by different routes, whereas the regeneration of acetyl-CoA from succinyl-CoA proceeds via the same route. Thermoproteales would be an exception to this unifying concept, since for Thermoproteus neutrophilus, the reductive citric acid cycle was proposed as a carbon fixation mechanism. Here, evidence is presented for the operation of the dicarboxylate/4-hydroxybutyrate cycle in this archaeon. All required enzyme activities were detected in large amounts. The key enzymes of the cycle were strongly upregulated under autotrophic growth conditions, indicating their involvement in autotrophic CO 2 fixation. The corresponding genes were identified in the genome.14 C-labeled 4-hydroxybutyrate was incorporated into the central building blocks in accordance with the key position of this compound in the cycle. Moreover, the results of previous 13 C-labeling studies, which could be reconciled with a reductive citric acid cycle only when some assumptions were made, were perfectly in line with the new proposal. We conclude that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO 2 fixation in the strict anaerobic Thermoproteales as well as in Desulfurococcales.Two new autotrophic carbon fixation cycles have recently been discovered in the Crenarchaea, one of the two subgroups of the Archaea. The 3-hydroxypropionate/4-hydroxybutyrate cycle functions in the aerobic autotrophic Sulfolobales (7) and the dicarboxylate/4-hydroxybutyrate cycle (Fig. 1) in the anaerobic autotrophic Ignicoccus hospitalis, belonging to the Desulfurococcales (27). These pathways have in common the synthesis of succinyl-coenzyme A (CoA) from acetyl-CoA and two inorganic carbons, although this is accomplished in quite different ways and using different carboxylases. In the 3-hydroxypropionate/4-hydroxybutyrate cycle, acetyl-CoA/propionyl-CoA carboxylase fixes two molecules of bicarbonate, and in the dicarboxylate/4-hydroxybutyrate cycle, pyruvate synthase and phosphoenolpyruvate (PEP) carboxylase are the two carboxylating enzymes. Yet, the regenerations of acetyl-CoA, the primary CO 2 acceptor, from succinyl-CoA are similar in the two pathways.Acetyl-CoA regeneration is as follows. The CO 2 fixation product succinyl-CoA is reduced to 4-hydroxybutyrate, which is activated to 4-hydroxybutyryl-CoA and then dehydrated to crotonyl-CoA by 4-hydroxybutyryl-CoA dehydratase. This radical [4Fe-4S] and flavin adenine dinucleotide-containing dehydratase (11, 37) is considered a key enzyme of the 4-hydroxybutyrate part of each pathway. Its product, crotonyl-Co...

Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota

“…The cells were harvested by centrifugation during the exponential growth phase and stored at 270 uC until use. Thermoproteus neutrophilus and M. sedula were grown autotrophically as reported previously (Alber et al, 2006;Ramos-Vera et al, 2009). …”

“…Pyruvate : water dikinase (EC 2.7.9.2), succinyl-CoA synthetase (EC 6.2.1.4, 6.2.1.5), 4-hydroxybutyrate-CoA ligase (EC 6.2.1.-), acetoacetyl-CoA b-ketothiolase (EC 2.3.1.9), malic enzyme [NAD(P)-dependent malate dehydrogenase (decarboxylating)] (EC 1.1.1.38, 1.1.1.40) were measured as described previously (Ramos-Vera et al, 2009). 3-Hydroxypropionate-CoA ligase (EC 6.2.1.-) was measured as described for 4-hydroxybutyrate-CoA ligase, but with 3-hydroxypropionate (10 mM) instead of 4-hydroxybutyrate as substrate.…”

“…The last two pathways were described recently in the (hyper)thermophilic autotrophic Crenarchaeota and, up to now, are restricted to this group of Archaea (Norris et al, 1989;Ishii et al, 1996;Burton et al, 1999;Menendez et al, 1999;Berg et al, 2007;Jahn et al, 2007;Huber et al, 2008;Ramos-Vera et al, 2009;Hügler et al, 2003a, b).…”

“…We provide evidence for the operation of the 3-hydroxypropionate/4-hydroxybutyrate cycle in S. azoricus and the dicarboxylate/4-hydroxybutyrate cycle in P. fumarii. Apparently, the oxygen-sensitive enzymes of the latter pathway operate at ; (b) the dicarboxylate/4-hydroxybutyrate cycle, as proposed for the Desulfurococcales and Thermoproteales (Huber et al, 2008;Ramos-Vera et al, 2009) high temperatures even under (micro)aerobic conditions. Therefore, the distribution of these two cycles follows a 16S rRNA-sequence-derived phylogenetic pattern.…”

“…All Crenarchaeota studied so far use either the 3-hydroxypropionate/4-hydroxybutyrate cycle or the dicarboxylate/4-hydroxybutyrate cycle, as has been experimentally shown for Metallosphaera sedula (Sulfolobales), Ignicoccus hospitalis (Desulfurococcaceae, Desulfurococcales) and Thermoproteus neutrophilus (Thermoproteales), and generalized based on whole-genome comparisons Jahn et al, 2007;Huber et al, 2008;Ramos-Vera et al, 2009). These pathways have in common the synthesis of succinyl-CoA from acetyl-CoA and two inorganic carbons, although this is accomplished in quite different ways and using different carboxylases (Fig.…”