Data_Sheet_2_Enzymes Catalyzing Crotonyl-CoA Conversion to Acetoacetyl-CoA During the Autotrophic CO2 Fixation in Metallosphaera sedula.xlsx

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.

自养泉古菌（Autotrophic Crenarchaeota）采用两种不同的二氧化碳固定循环。硫化叶菌目（Sulfolobales）成员采用3-羟基丙酸/4-羟基丁酸（3-hydroxypropionate/4-hydroxybutyrate, HP/HB）循环，而脱硫球菌目（Desulfurococcales）和热变形菌目（Thermoproteales）则采用二羧酸/4-羟基丁酸循环。
尽管这两种循环在将乙酰辅酶A（acetyl-CoA）+ 2 CO₂转化为琥珀酰辅酶A（succinyl-CoA）的羧化反应步骤中存在差异，但二者拥有共同的再生阶段：即琥珀酰辅酶A重新转化为两分子乙酰辅酶A的过程。该共同阶段包含巴豆酰辅酶A（crotonyl-CoA）转化为乙酰乙酰辅酶A（acetoacetyl-CoA）的步骤，该反应已在医院伊格尼球菌（Ignicoccus hospitalis，隶属于脱硫球菌目）和嗜中性热棒菌（Pyrobaculum neutrophilus，隶属于热变形菌目）中被明确证实由双功能巴豆酸酶/3-羟基丁酰辅酶A脱氢酶（bifunctional crotonase/3-hydroxybutyryl-CoA dehydrogenase）催化。
该酶为融合蛋白，包含烯酰辅酶A水合酶（enoyl-CoA hydratase）与脱氢酶结构域两个组分。由于硫化叶菌目中同样存在该同源双功能蛋白，因此学界曾提出其在巴豆酰辅酶A向乙酰乙酰辅酶A的转化过程中发挥普遍功能的假说。
本研究表明，作为硫化叶菌目自养模式物种的金属硫化叶菌（Metallosphaera sedula），除了该双功能蛋白（Msed_0399）之外，还携带多个分别编码巴豆酰辅酶A水合酶（crotonyl-CoA hydratase）和（S）-3-羟基丁酰辅酶A脱氢酶（(S)-3-hydroxybutyryl-CoA dehydrogenase）的独立基因。此前已有研究证实，这些基因在自养与混养条件下均可发生转录。
脱氢酶Msed_1423（而非双功能蛋白Msed_0399）似乎是催化（S）-3-羟基丁酰辅酶A脱氢反应的核心酶类。该脱氢酶的同源蛋白是所有自养硫化叶菌目中唯一存在的（S）-3-羟基丁酰辅酶A脱氢酶，这进一步强化了上述结论。
金属硫化叶菌基因组中存在两个尚未表征的巴豆酸酶同源蛋白（Msed_0336与Msed_0384），我们对其进行了异源重组表达并完成功能表征。两种蛋白均为高效的巴豆酰辅酶A水合酶，可能在体内HP/HB循环的对应反应中发挥功能（或直接负责该反应）。