Abstract
Autotrophic Crenarchaeota use two different cycles for carbon dioxide fixation. Members of the Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, whereas Desulfurococcales and Thermoproteales use the dicarboxylate/4-hydroxybutyrate cycle. While these two cycles differ in the carboxylation reactions resulting in the conversion of acetyl-CoA + 2 CO2 to succinyl-CoA, they have a common regeneration part in which succinyl-CoA is reconverted to two acetyl-CoA molecules. This common part includes crotonyl-CoA conversion to acetoacetyl-CoA, which has unequivocally been shown in Ignicoccus hospitalis (Desulfurococcales) and Pyrobaculum neutrophilus (Thermoproteales) to be catalyzed by a bifunctional crotonase/3-hydroxybutyryl-CoA dehydrogenase. It is a fusion protein consisting of an enoyl-CoA hydratase and a dehydrogenase domain. As the homologous bifunctional protein is present in Sulfolobales as well, its common functioning in the conversion of crotonyl-CoA to acetoacetyl-CoA was proposed. Here we show that a model autotrophic member of Sulfolobales, Metallosphaera sedula, possesses in addition to the bifunctional protein (Msed_0399) several separate genes coding for crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase. Their genes were previously shown to be transcribed under autotrophic and mixotrophic conditions. The dehydrogenase Msed_1423 (and not the bifunctional protein Msed_0399) appears to be the main enzyme catalyzing the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Homologs of this dehydrogenase are the only (S)-3-hydroxybutyryl-CoA dehydrogenases present in all autotrophic Sulfolobales, strengthening this conclusion. Two uncharacterized crotonase homologs present in M. sedula genome (Msed_0336 and Msed_0384) were heterologously produced and characterized. Both proteins were highly efficient crotonyl-CoA hydratases and may contribute (or be responsible) for the corresponding reaction in the HP/HB cycle in vivo.
Highlights
Autotrophic CO2 fixation is the most important biosynthetic process in nature, being responsible for the primary production of organic matter
Genome analysis shows that M. sedula and most other Sulfolobales have multiple homologs of genes responsible for the conversion of crotonyl-CoA into acetyl-CoA, and our data suggest that crotonyl-CoA hydratase and (S)-3-hydroxybutyrylCoA dehydrogenase reactions may be catalyzed by several proteins in vivo
Analysis of deletion mutants would be very helpful to clarify the role of individual proteins in autotrophic CO2 fixation, genetic system is not available for autotrophic Sulfolobales
Summary
Autotrophic CO2 fixation is the most important biosynthetic process in nature, being responsible for the primary production of organic matter. Several fundamentally different CO2 fixation pathways evolved. They vary in their reaction sequences, and in their types of carboxylases, cofactors, and electron donors used to fix inorganic carbon into biomass (Berg et al, 2010a; Berg, 2011; Fuchs, 2011; Hügler and Sievert, 2011). A formally similar, but phylogenetically unrelated pathway was shown in aerobic ammonia-oxidizing Thaumarchaeota (Könneke et al, 2014; Otte et al, 2015). While the autotrophic cycle in Thaumarchaeota is less studied, with some characteristic enzymes of the cycle still being unidentified, all enzymes of the crenarchaeal cycle were identified and biochemically characterized (Table 1)
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