3-hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenzyme A reductase, enzymes of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in the Sulfolobales

Abstract

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO(2) fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.

FIG. 1.

Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other members of the Sulfolobales. Enzymes are the following: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionyl-CoA synthetase (3-hydroxypropionate-CoA ligase, AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinate semialdehyde reductase (NADPH); 12, 4-hydroxybutyryl-CoA synthetase (4-hydroxybutyrate-CoA ligase, AMP-forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The two steps of interest are highlighted.

FIG. 2.

SDS-PAGE (12.5%) of fractions obtained during purification of native and recombinant acryloyl-CoA reductase. Proteins were stained with Coomassie blue. (A) Enzyme fractions during purification of the native enzyme from M. sedula. Lane 1, cell extract of autotrophically grown cells (20 μg); lane 2, after heat precipitation (20 μg); lane 3, after ultracentrifugation (20 μg); lane 4, after Q-Sepharose chromatography (20 μg); lane 5, after carboxymethylcellulose chromatography (20 μg); lane 6, after phenyl-Sepharose chromatography (10 μg; after an additional purification step using a Resource S column the marked band was cut out and sequenced); lane 7, molecular mass standard proteins. (B) Heterologous expression of the acryloyl-CoA reductase gene from S. tokodaii in E. coli Rosetta 2 (DE3). Lane 1, whole cells before induction; lane 2, whole cells after 3 h of induced growth; lane 3, cell extract after heat precipitation (20 μg); lane 4, purified recombinant acryloyl-CoA reductase after Ni²⁺ affinity column (20 μg); lane 5, molecular mass standard proteins.

FIG. 3.

SDS-PAGE (12.5%) of fractions obtained during purification of native and recombinant 3-hydroxypropionyl-CoA dehydratase. Proteins were stained with Coomassie blue. (A) Enzyme fractions during purification of the native enzyme from M. sedula. Lane 1, cell extract of autotrophically grown cells (20 μg); lane 2, after heat precipitation (20 μg); lane 3, after dialysis overnight (20 μg); lane 4, after Q-Sepharose chromatography (10 μg); lane 5, after carboxymethylcellulose chromatography (10 μg); lane 6, after Cibacron blue chromatography (the marked band is 3-hydroxypropionyl-CoA dehydratase; protein amount not determined); lane 7, molecular mass standard proteins. (B) Heterologous expression of the 3-hydroxypropionyl-CoA dehydratase gene from M. sedula in E. coli Rosetta 2 (DE3). Lane 1, whole cells before induction; lane 2, whole cells after 3 h of induced growth; lane 3, cell extract after heat precipitation (20 μg); lane 4, purified recombinant 3-hydroxypropionyl-CoA dehydratase after Ni²⁺ affinity column (10 μg); lane 5, molecular mass standard proteins.

FIG. 4.

Phylogenetic tree of acryloyl-CoA reductase. The tree is based on amino acid sequence analysis and rooted with propionyl-CoA synthase from C. aurantiacus. Tree topography and evolutionary distances are given by the neighbor-joining method with Poisson correction. The scale bar represents a difference of 0.1 substitutions per site. Numbers at nodes indicate the percentage bootstrap values for the clade of this group in 1,000 replications. The enzyme group from the order Sulfolobales is indicated.

FIG. 5.

Phylogenetic tree of 3-hydroxypropionyl-CoA dehydratase. The tree is based on amino acid sequence analysis and rooted with propionyl-CoA synthase from C. aurantiacus. Tree topography and evolutionary distances are given by the neighbor-joining method with Poisson correction. The scale bar represents a difference of 0.1 substitutions per site. Numbers at nodes indicate the percentage bootstrap values for the clade of this group in 1,000 replications. The enzyme groups from the order Sulfolobales and from Clostridia are indicated. Fusion refers to the dehydratase domain of an archaeal fusion enzyme that contains an enoyl-CoA hydratase fused to a 3-hydroxyacyl-CoA dehydrogenase.