Chemicals and biochemicals were obtained from Fluka (Neu-Ulm, Germany), Sigma-Aldrich (Deisenhofen, Germany), Merck (Darmstadt, Germany), VWR (Darmstadt, Germany), Roth (Karlsruhe, Germany), Applichem (Darmstadt, Germany), IBA (Göttingen, Germany) or Cell Signaling Technology (Frankfurt, Germany). Materials for cloning and expression were purchased from MBI Fermentas (St. Leon-Rot, Germany), New England Biolabs (Frankfurt, Germany), and Novagen (Schwalbach, Germany). Materials and equipment for protein purification were obtained from GE Healthcare (Freiburg, Germany), Macherey-Nagel (Düren, Germany), Pall Corporation (Dreieich, Germany) or Millipore (Eschborn, Germany). Primers were synthesized by Sigma-Aldrich (Steinheim, Germany).

Crotonyl-CoA and methacrylyl-CoA were synthesized from the corresponding anhydrides and CoA by the method of Simon and Shemin (1953). (S)-3-hydroxybutyryl-CoA, (R)-3-hydroxybutyryl-CoA, and (E)-2-octenoyl-CoA were synthesized chemically from the corresponding free acids by the mixed anhydride method described by Stadtman (1957). 3-Hydroxypropionyl-CoA was synthesized enzymatically with recombinant propionate CoA-transferase from Clostridium propionicum as described by Selmer et al. (2002). Acryloyl-CoA was synthesized enzymatically with recombinant acyl-CoA oxidase 4 from Arabidopsis thaliana as described in Schwander et al. (2016). Crotonyl-CoA, 3-hydroxybutyryl-CoA, and (E)-2-octenoyl-CoA were purified using HPLC (Zarzycki et al., 2009). The dry powders of the CoA esters were stored at −20°C.

Cells of M. sedula TH2^T (DSM 5348) were grown autotrophically at 75°C modified Allen medium, pH 2.0 (Huber and Prangishvili, 2006) in a 100-l fermenter under gassing with a mixture of 19% CO₂, 3% O₂, and 78% H₂ (0.5 l/min) (generation time, 8 h) (Huber et al., 1989, 1992). Cells were frozen in liquid nitrogen and stored at −80°C until use. Escherichia coli strain DH5α and E. coli strain Rosetta 2 (DE3) (Merck, Germany) were grown at 37°C in lysogeny broth (LB) medium. Antibiotics were added to the cultures to a final concentration of 100 μg ampicillin ml^–1 and 34 μg chloramphenicol ml^–1.

Frozen cells were suspended in a double volume of 20 mM Tris–HCl (pH 7.8) containing 0.1 mg ml^–1 DNase I. The cell suspensions were passed twice through a chilled French pressure cell at 137 MPa, and the cell lysates were centrifuged for 1 h (100,000 × g; 4°C). The supernatant (cell extract) was used for protein purification or enzyme assays immediately.

Standard protocols were used for purification, cloning, transformation, and amplification of DNA (Ausubel et al., 1987). Plasmid DNA was isolated with the Monarch Plasmid Miniprep Kit (NEB). Primers and restriction enzymes used for the cloning of M. sedula genes are listed in Supplementary Table S1. The genes were amplified using Q5 polymerase (NEB). PCR conditions were as follows: 35 cycles of 10-s denaturation at 98°C, 30-s primer annealing at 60°C and 30-s elongation at 72°C. The PCR products were treated with the corresponding restrictases (Supplementary Table S1), and the genes were separately ligated into the expression vector pET16b containing a sequence encoding an N-terminal His₁₀-tag. The plasmids were transformed into E. coli DH5α for amplification, followed by their purification and sequencing.

The amplified expression vectors were used to transform E. coli Rosetta 2 (DE3). The cells were grown at 37°C in LB medium with ampicillin and chloramphenicol. Expression was induced at an optical density (OD_{578 nm}) of 0.6 with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG), and the temperature was lowered to 20°C. The cells were harvested after additional growth for 4 h and stored at −20°C until use.

Recombinant crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase Msed_0399 was produced untagged. The protein was purified as described previously (Ramos-Vera et al., 2011). The heterologously produced His₁₀-tagged (S)-3-hydroxybutyryl-CoA dehydrogenases Msed_1423 and Msed_0389 as well as crotonyl-CoA hydratases Msed_0336 and Msed_0384 were purified using affinity chromatography. The corresponding cell extracts were applied at a flow rate of 0.5 ml min^–1 to a 1-ml Protino Ni-NTA column (Macherey-Nagel) that had been equilibrated with 20 mM Tris–HCl containing 150 mM KCl (pH 7.8 for Msed_1423 and Msed_0389, pH 7.6 for Msed_0384, and pH 8.0 for Msed_0336). The column was washed with the same buffer containing 50 mM imidazole at a flow rate of 0.5 ml min^–1 to elute unwanted protein. The recombinant enzymes were eluted with the same buffer containing 500 mM imidazole. The enzymes were concentrated using 10K Vivaspin Turbo 4 (Sartorius, Göttingen, Germany) and stored at −20°C with 50% glycerol. The identity of the purified recombinant proteins was confirmed using gel digestion by trypsin followed by LC-MS/MS (Supplementary Table S2).

The 3-hydroxybutyryl-CoA dehydratase and 3-hydroxypropionyl-CoA dehydratase activities were measured at 42°C. All other enzyme activities were measured at 65°C.

CoA and CoA-esters were detected with Agilent 1290 Infinity II UHPLC using a reversed-phase C18 column (Agilent InfinityLab Poroshell 120 EC-C18 1.9 μm 2.1 × 50 mm column). The following acetonitrile gradient in 10 mM potassium phosphate buffer (pH 7), with a flow rate of 0.55 ml min^–1, was used: from 2 to 8% at 0–2.66 min; from 8 to 30% at 2.66–3.33 min; from 30 to 2% at 3.33–3.68 min; 2% at 3.68–5 min. Retention times were: 2-methylmalonyl-CoA, 0.9 min; 3-hydroxypropionyl-CoA, 1.4 min; acetyl-CoA, 1.6 min; 3-hydroxybutyryl-CoA, 1.8 min; 3-hydroxy-2-methylpropionyl-CoA, 2.0 min; acryloyl-CoA, 2.2 min; propionyl-CoA, 2.4 min; crotonyl-CoA, 2.6 min; methacrylyl-CoA, 2.9 min. For the conversions with (E)-2-octenoyl-CoA, the following acetonitrile gradient in 10 mM potassium phosphate buffer (pH 6.8), with a flow rate of 0.55 ml min^–1, was used: from 2 to 30% at 0–3.33 min; from 30 to 2% at 3.33–3.68 min; 2% at 3.68–5 min. Retention times were: 3-hydroxyoctanoyl-CoA, 2.3 min; 3-oxo-octanoyl-CoA, 2.4 min; (E)-2-octenoyl-CoA, 3.2 min. Reaction products and standard compounds were detected by UV absorbance at 260 nm with 1290 Infinity II diode array detector (Agilent), and the amount of product was calculated from the relative peak area. The identification of the CoA esters was based on co-chromatography with standards and analysis of the UV spectra of the products.

Apparent K_m and V_max values were calculated using GraphPad Prism5 software. Protein concentration was determined using the Bradford method (Bradford, 1976) with bovine serum albumin as a standard. DNA sequence determination was performed by Eurofins (Ebersberg, Germany). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12.5%) was performed as described by Laemmli (1970). An unstained protein MW marker (Thermo Scientific, 14.4-116 kDa) and a prestained protein marker (Cell Signaling, 11-190 kDa) were used as molecular mass standard. Proteins were visualized by Coomassie blue staining (Zehr et al., 1989). Protein identification was performed at the IZKF Core Unit Proteomics Münster based on tryptic in-gel digestion and mass spectrometric analysis using Synapt G2 Si coupled to M-Class (Waters Corp.). Query sequences for the database searches were obtained from the National Center for Biotechnology Information (NCBI) data base. The BLAST searches were performed via NCBI BLAST server¹ (Altschul et al., 1990).

Msed_0399 is a bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase consisting of two domains, a C-terminal enoyl-CoA hydratase domain (∼20 kDa) and an N-terminal dehydrogenase domain (∼40 kDa) (Ramos-Vera et al., 2011). This enzyme is widely distributed among Archaea and has been predicted to be present in the common ancestor of the TACK superphylum, i.e., it was already present in the common ancestor of Cren- and Thaumarchaeota (Williams et al., 2017). The enzyme was purified formerly from autotrophically grown M. sedula cells, following the reduction of NAD with crotonyl-CoA, i.e., the presence of both crotonase and dehydrogenase reactions simultaneously (Ramos-Vera et al., 2011). The identified msed_0399 was cloned and heterologously produced, and the recombinant enzyme was characterized (Ramos-Vera et al., 2011; Hawkins et al., 2014). However, the characterization was preliminary, as the activity was measured only as crotonyl-CoA or (S)-3-hydroxybutyryl-CoA-dependent NAD reduction (Ramos-Vera et al., 2011; Hawkins et al., 2014). The measured hydratase activity in the coupled assay was necessarily limited by the level of the much lower dehydrogenase activity.

In order to fill this gap, we heterologously produced and purified the bifunctional protein Msed_0399, and characterized the enzyme by measuring dehydrogenase and crotonase reactions separately. In hydratase reaction, Msed_0399 was promiscuous and could hydrate not only crotonyl-CoA, but also (E)-2-octenoyl-CoA and acrylyl-CoA (Table 2). Similarly, it acted as dehydrogenase with both C₄ and C₈ (S)-3-hydroxyacyl-CoAs (Table 2). Interestingly, the enzyme was most active with octenoyl-CoA, thus suggesting that Msed_0399 may participate in β-oxidation, in addition to autotrophic CO₂ fixation. Importantly, Msed_0399 is the only 3-hydroxyacyl-CoA dehydrogenase that was active with C₈ (3-hydroxy)acyl-CoAs (Tables 2, 3), further suggesting its participation in heterotrophic metabolism.

The apparent K_m value of Msed_0399 for crotonyl-CoA in the crotonase/(S)-3-hydroxybutyryl-CoA dehydrogenase reaction was four times higher than the corresponding value in M. sedula cell extract (Table 2) suggesting the presence of additional crotonase(s). Furthermore, the apparent K_m value of Msed_0399 for (S)-3-hydroxybutyryl-CoA in the dehydrogenase reaction was three times higher than the corresponding value in M. sedula cell extract (Table 2). These data suggest that Msed_0399 may not be the only enzyme responsible for crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase activity in autotrophically grown M. sedula. Therefore, we cloned and expressed other candidate genes for these enzymes (Table 1) in E. coli and characterized the catalytic activities of the resulting enzymes.

The purified recombinant Msed_0389 and Msed_1423 catalyzed the NAD-dependent oxidation of (S)-3-hydroxybutyryl-CoA to acetoacetyl-CoA and were highly specific for their substrate (K_m 2.6 and 5 μM, respectively; Table 3). Both enzymes were not active with (S)-3-hydroxyoctanoyl-CoA. Their k_cat/K_m values were 3- and 20-fold higher than the corresponding value of Msed_0399 in (S)-3-hydroxybutyryl-CoA dehydrogenase reaction (Table 3). Interestingly, both these genes were shown to be up-regulated in auto- and mixotrophically grown cell (see Table 1; Auernik and Kelly, 2010; Ai et al., 2019). The Msed_0389 gene clustered together with various β-oxidation genes in the M. sedula genome. In contrast, msed_1423 clustered with several genes encoding specific enzymes of the HP/HB cycle, suggesting its involvement in autotrophic CO₂ fixation (Figure 2).

The purified recombinant Msed_0336 and Msed_0384 catalyzed the hydration of crotonyl-CoA to (S)-3-hydroxybutyryl-CoA (Table 4); the k_cat/K_m values of both enzymes for the reverse reaction were ∼2.5 times higher. Both enzymes were also active with (E)-2-octenoyl-CoA and, to a lesser extent, with acrylyl-CoA and methacrylyl-CoA. The V_max values of these enzymes for crotonyl-CoA hydration were lower than the V_max of Msed_0399; yet their apparent crotonyl-CoA K_m values were also much lower, resulting in a catalytic constant k_cat/K_m similar to that of Msed_0399. The clustering of msed_0384 with β-oxidation genes conforms with its higher k_cat/K_m value for octenoyl-CoA. Nevertheless, it is Msed_0399 which has the highest k_cat/K_m value for this substrate. In the published transcriptomic analyses, Msed_0384 was shown to be down-regulated in auto- and mixotrophically grown cell, while Msed_0336 showed no significant regulation in these cells (see Table 1; Auernik and Kelly, 2010; Ai et al., 2019).

The genes of the HP/HB cycle catalyzing reactions from acetyl-CoA to crotonyl-CoA are present in all fully sequenced Sulfolobales genomes (Supplementary Table S3), indicating that they have the potential to grow autotrophically. The only exception was Sulfodiicoccus acidiphilus that does not possess most of the key genes of the HP/HB cycle. This species is only distantly related to other Sulfolobales and is not capable to grow autotrophically (Sakai and Kurosawa, 2017, 2019). On the contrary, most of the other Sulfolobales are autotrophs, with the exception of Sulfolobus acidocaldarius and Saccharolobus solfataricus that nevertheless encode all key genes of the HP/HB cycle in their genomes (Supplementary Table S3).

Homolog(s) of bifunctional crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase Msed_0399 genes were found in 75% of the genomes, whereas four autotrophic species (Acidianus ambivalens, Acidianus hospitalis, Acidianus sulfidivorans, and Stygiolobus azoricus) do not possess the corresponding gene (Table 5). Notably, the functioning of the HP/HB cycle was experimentally shown for S. azoricus (Berg et al., 2010b), indicating that the presence of the bifunctional protein is not essential for autotrophic CO₂ fixation. The only two genes that are (almost) universally distributed in Sulfolobales are the crotonase Msed_0566 gene and the dehydrogenase Msed_1423 gene (Table 5 and Supplementary Table S3). They are absent only in the genome of S. acidocaldarius.