Enzymes Catalyzing Crotonyl-CoA Conversion to Acetoacetyl-CoA During the Autotrophic CO2 Fixation in Metallosphaera sedula

Li Liu; Harald Huber; Ivan A Berg

doi:10.3389/fmicb.2020.00354

Enzymes Catalyzing Crotonyl-CoA Conversion to Acetoacetyl-CoA During the Autotrophic CO₂ Fixation in Metallosphaera sedula

Front Microbiol. 2020 Mar 11:11:354. doi: 10.3389/fmicb.2020.00354. eCollection 2020.

Authors

Li Liu¹, Harald Huber², Ivan A Berg¹

Affiliations

¹ Institute for Molecular Microbiology and Biotechnology, University of Münster, Münster, Germany.
² Institute for Microbiology and Archaeal Center, University of Regensburg, Regensburg, Germany.

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.

Keywords: 3-hydroxybutyryl-CoA dehydrogenase; 3-hydroxypropionate/4-hydroxybutyrate cycle; Metallosphaera sedula; Sulfolobales; crotonyl-CoA hydratase.