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J Bacteriol. 2009 October; 191(20): 6352–6362.
Published online 2009 August 14. doi:  10.1128/JB.00794-09
PMCID: PMC2753041

Malonic Semialdehyde Reductase, Succinic Semialdehyde Reductase, and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales[down-pointing small open triangle]

Abstract

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO2 fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.

The thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula uses a 3-hydroxypropionate/4-hydroxybutyrate cycle for CO2 fixation (9, 28, 29, 35) (Fig. (Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales (31) and in mesophilic marine group I Crenarchaea (Cenarchaeum sp., Nitrosopumilus sp.). This cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (15, 22-25, 41, 42). It involves the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA by a biotin-dependent acetyl-CoA carboxylase (12, 29). The carboxylation product is reduced to malonic semialdehyde by malonyl-CoA reductase (1). Malonic semialdehyde is further reduced to 3-hydroxypropionate, the characteristic intermediate of the pathway (9, 31, 35). 3-Hydroxypropionate is further reductively converted to propionyl-CoA (3), which is carboxylated to (S)-methylmalonyl-CoA by propionyl-CoA carboxylase. Only one copy of the genes encoding the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, indicating that this enzyme is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (12, 29). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, which is followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase.

FIG. 1.
Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other autotrophic Sulfolobales. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP ...

Succinyl-CoA is converted via succinic semialdehyde and 4-hydroxybutyrate to two molecules of acetyl-CoA (9), thus regenerating the starting CO2 acceptor molecule and releasing another acetyl-CoA molecule for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. (Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA molecule and two bicarbonate molecules into succinyl-CoA (Fig. (Fig.1,1, steps 1 to 9), and the second part converts succinyl-CoA to two acetyl-CoA molecules (Fig. (Fig.1,1, steps 10 to 16).

The second part of the autotrophic cycle also occurs in the dicarboxylate/4-hydroxybutyrate cycle, which operates in autotrophic CO2 fixation in Desulfurococcales and Thermoproteales (Crenarchaea) (27, 37), raising the question of whether the enzymes in these two lineages have common roots (37). The first part of the cycle also occurs in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus and a few related green nonsulfur phototrophic bacteria (19, 22, 23, 32, 49).

The two-step reduction of malonyl-CoA to 3-hydroxpropionate in Chloroflexus is catalyzed by a single bifunctional 300-kDa enzyme (30). The M. sedula malonyl-CoA reductase is completely unrelated and forms only malonic semialdehyde (1), and the enzyme catalyzing the second malonic semialdehyde reduction step that forms 3-hydroxypropionate is unknown. In the second part of the 3-hydroxypropionate/4-hydroxybutyrate cycle a similar reduction of succinyl-CoA via succinic semialdehyde to 4-hydroxybutyrate takes place. The enzymes responsible for these reactions also have not been characterized.

In this work we purified the enzymes malonic semialdehyde reductase, succinyl-CoA reductase, and succinic semialdehyde reductase from M. sedula. The genes coding for these enzymes were identified in the genome, and recombinant proteins were studied in some detail. Interestingly, succinyl-CoA reductase turned out to be identical to malonyl-CoA reductase. We also show here that enzymes that are highly similar to succinyl-CoA reductase in Thermoproteus neutrophilus do not function as succinyl-CoA reductases in M. sedula.

MATERIALS AND METHODS

Materials.

Chemicals and biochemicals were obtained from Roche Diagnostics (Mannheim, Germany), Fluka (Neu-Ulm, Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), Sigma-Aldrich (Deisenhofen, Germany), Bio-Rad (München, Germany), and Genaxxon (Biberach, Germany). Gases were obtained from Sauerstoffwerke Friedrichshafen (Friedrichshafen, Germany). Enzymes and primers were obtained from MBI Fermentas (St. Leon-Rot, Germany) and Genaxxon Biosciences GmbH (Biberach, Germany). Materials and equipment used for protein purification were obtained from GE Healthcare (München, Germany), Millipore (Bedford, MA), Sigma-Aldrich (Deisenhofen, Germany), and Whatman Biosystems Ltd. (Madistone, Great Britain). Plasmids were obtained from Invitrogen (Karlsruhe, Germany), Novagene (Darmstadt, Germany), and Fermentas (St. Leon-Rot, Germany).

Strains and culture conditions.

M. sedula TH2 (= DSM 5348) was grown autotrophically at 75°C on a chemically defined medium (pH 2.0) with a gas mixture containing 19% CO2, 3% O2, and 78% H2, and the generation time was 20 h (26). Control cells were grown (micro)aerobically and heterotrophically with 0.05% yeast extract with a generation time of 16 h (26). Escherichia coli strains DH5α and Rosetta 2(DE3) (Merck, Germany) were grown at 37°C in lysogeny broth (39). Antibiotics were added to E. coli cultures at the following final concentrations: 100 μg ampicillin ml−1 and 34 μg chloramphenicol ml−1. Cells were stored frozen in liquid nitrogen until they were used.

Enzyme assays.

The enzyme activities were determined at 65°C (growth temperature, 75°C) by spectrophotometric assays (volume of the assay mixture, 0.5 ml), in which the substrate-dependent oxidation of NAD(P)H or the reduction of NAD(P)+ was followed spectrophotometrically at 365 nm [epsilon at 365 nm for NAD(P)H, 3,400 M−1 cm−1]. When NAD(P)H concentrations higher than 0.5 mM were used, a cuvette with a 0.1-cm light path was used. None of the enzymes was oxygen sensitive. The pH of the buffers was adjusted at 20°C. The actual pH at 65°C could be extrapolated using previously published dpKa/dT values indicating the pKa shift per 1°C increase in the temperature (dpKa/dT values were obtained from references 13, 16, and 20); pKa values for 20°C were obtained from references 18, 20, and 21). The buffers used to determine the optimum pH were 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH (pH 6.4 to 6.9; dpKa/dT, −0.011), 3-(N-morpholino)propanesulfonic acid (MOPS)-NaOH (pH 7.2 to 8.4; dpKa/dT, −0.011), B(OH)3-HCl (pH 9.3; dpKa/dT, −0.008), Tris-HCl (pH 9.1 to 10.1; dpKa/dT, −0.028), and 2-aminoethanol-HCl (pH 10.5 to 11; dpKa/dT, −0.025). To test the effect of metal-chelating agents, protein fractions were incubated for 16 h on ice with 0, 5, and 10 mM EDTA or for 1 h at 30°C and 65°C without and with 100 mM EDTA (34), and the assay mixture contained 10 mM EDTA.

(i) Malonic semialdehyde reductase.

Malonic semialdehyde was enzymatically synthesized from malonyl-CoA using recombinant malonyl-CoA reductase from Sulfolobus tokodaii (1). The malonic semialdehyde-dependent oxidation of NADPH was followed. The assay mixture contained 100 mM MOPS-KOH (pH 7.4), 5 mM MgCl2, 5 mM 1,4-dithioerythritol, 0.5 mM NADPH, 1 to 3 U (1 to 3 μmol min−1) of recombinant malonyl-CoA reductase, and 0.2 mM malonyl-CoA. After 5 min of incubation to allow formation of malonic semialdehyde, the reaction was started by adding extract or (partially) purified enzyme. For determination of the apparent Km the concentration of one substrate was varied (0.025 to 0.5 mM malonic semialdehyde; 0.017 to 0.45 mM NADPH) while the concentration of the other substrate was kept constant (0.3 mM malonic semialdehyde; 0.5 mM NADPH, kept constant by addition of NADPH after malonyl-CoA reduction was complete).

(ii) Succinic semialdehyde reductase.

The assay mixture contained 100 mM MOPS-NaOH (pH 7.9), 0.5 mM NADPH, 0.2 mM succinic semialdehyde, and cell extract or (partially) purified enzyme. The reaction was started by addition of succinic semialdehyde. For determination of the apparent Km, the concentration of one substrate was varied (0.01 to 0.5 mM succinic semialdehyde; 0.015 to 0.5 mM NADPH), while the concentration of the other substrate was kept constant (1 mM succinic semialdehyde; 0.5 mM NADPH).

(iii) Succinyl-CoA reductase.

The assay mixture contained 100 mM Tris-HCl (pH 7.8), 5 mM MgCl2, 5 mM 1,4-dithioerythritol, 0.5 mM NADPH, 0.2 mM succinyl-CoA, and cell extract or (partially) purified enzyme. The reaction was started by addition of succinyl-CoA. For determination of the apparent Km the concentration of one substrate was varied (0.05 to 3 mM succinyl-CoA; 0.05 to 2 mM NADPH), while the concentration of the other substrate was kept constant (1.5 mM succinyl-CoA; 0.5 mM NADPH). Succinyl-CoA was synthesized from its anhydride by a slightly modified method described by Simon and Shemin (40).

(iv) Succinic semialdehyde dehydrogenase.

The assay mixture contained 100 mM MOPS-NaOH (pH 8.1), 2.5 mM NAD(P)+, 2 mM succinic semialdehyde, and partially purified enzyme after heat precipitation. The reaction was started by addition of succinic semialdehyde. CoA (0.5 mM) was added in controls to detect a potential CoA-acylating succinic semialdehyde dehydrogenase.

(v) Malate dehydrogenase.

The assay mixture contained 100 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.5 mM NADH, and cell extract. The reaction was started by addition of 5 mM oxaloacetate.

Purification of enzymes.

Enzymes were purified at 8°C, unless otherwise stated. The pH of the buffers was adjusted at 20°C. Cell extracts were prepared by suspending 1 part of frozen cells in 1 to 2 parts of 10 mM Tris-HCl (pH 8.0) (designated buffer A for M. sedula cells) or 20 mM Tris-HCl (pH 8.0) (designated buffer B for E. coli cells) containing 5 μg DNase I ml−1 and were passed twice through a chilled French pressure cell at 137 MPa. The cell lysate was centrifuged at 100,000 × g for 1 h at 4°C, and the supernatant was considered the cell extract. For the heat precipitation steps temperatures at which the enzymes were stable were used.

(i) Malonic semialdehyde reductase.

The enzyme was purified from 11.5 g (wet mass) of frozen autotrophically grown M. sedula cells.

(a) DEAE-Sepharose chromatography.

Cell extract (15 ml) was applied to a DEAE-Sepharose column (fast flow; diameter, 1.6 cm; volume, 18 ml; GE Healthcare) that had been equilibrated with buffer A at a flow rate of 3 ml min−1. The column was washed with 4 bed volumes of buffer A and eluted with a step gradient of 100 mM KCl in buffer A. The activity eluted at 100 mM KCl. The most active fraction (12 ml) was kept frozen at −20°C in 30% glycerol until it was used.

(b) Q-Sepharose chromatography.

The enzyme fraction obtained from the DEAE-Sepharose chromatography step was loaded on a Q-Sepharose column (fast flow; diameter, 1.6 cm; volume, 18 ml; GE Healthcare) equilibrated with buffer A. The column was washed with buffer A and developed with a step gradient of 50 mM KCl in buffer A at a flow rate of 2 ml min−1. The active protein eluted at 50 mM KCl. The most active fractions were pooled (85 ml) and kept frozen at −20°C with 30% glycerol until they were used.

(c) Reactive green chromatography.

Three milliliters of the enzyme fraction obtained from the Q-Sepharose chromatography step was applied at room temperature to a 0.4-ml reactive green 19 column (Sigma-Aldrich) equilibrated with buffer B. The column was washed with 5 ml buffer B and 4 ml 200 mM KCl in buffer B. The activity was eluted with an NADPH step gradient (30, 40, 50, 100 μM and 1 mM NADPH) in buffer B; the highest activity eluted at 100 μM NADPH.

(ii) Recombinant malonic semialdehyde reductase.

All purification steps were done at room temperature. The enzyme was purified from 1 g (wet mass) of E. coli cells.

(a) Heat precipitation.

Extract (0.5 ml) was incubated at 70°C for 15 min and then cooled on ice for 15 min, and this was followed by centrifugation at 20,000 × g and 4°C for 15 min.

(b) Reactive green chromatography.

The supernatant obtained after heat precipitation was diluted with buffer B and applied to a 0.4-ml reactive green 19 column (Sigma-Aldrich) equilibrated with buffer B. The column was washed with 5 ml buffer B and 5 ml 200 mM KCl in buffer B. The enzyme was eluted with 1 ml of 0.25 mM NADPH in buffer B. The protein was stored in 20% glycerol at −20°C.

(iii) Succinic semialdehyde reductase.

The enzyme was prepared from 3.3 g (wet mass) of autotrophically grown M. sedula cells.

(a) DEAE-Sepharose chromatography.

Cell extract (5 ml) was applied to a DEAE-Sepharose column (fast flow; diameter, 1.6 cm; volume, 5 ml; GE Healthcare) that had been equilibrated with 10 mM Tris-HCl (pH 7.8) (designated buffer C) at a flow rate of 2 ml min−1. The column was washed with 5 bed volumes of buffer C and eluted with a step gradient of 50 mM KCl in buffer C. The activity eluted at 50 mM KCl. The most active fraction (5 ml) was kept frozen at −20°C.

(b) Phenyl-Sepharose chromatography.

A saturated ammonium sulfate solution was added to 4 ml of the active fraction obtained from the DEAE-Sepharose chromatography step to obtain a final concentration of 1 M. After centrifugation the supernatant was directly applied to a phenyl-Sepharose HR5/5 column (GE Healthcare) equilibrated with 100 mM Tris-HCl (pH 7.8)-1 M (NH4)2SO4 (designated buffer D) at a flow rate of 0.5 ml min−1. After the column was washed with equilibration buffer D, it was developed with decreasing (NH4)2SO4. The enzyme eluted with 800 mM (NH4)2SO4.

(iv) Recombinant succinic semialdehyde reductase.

The enzyme was purified from 1.5 g (wet mass) of E. coli cells.

(a) Heat precipitation.

Extract (3 ml) was incubated for 15 min at 75°C and then cooled on ice for 15 min, and this was followed by centrifugation at 20,000 × g and 4°C for 15 min.

(b) MonoQ-Sepharose chromatography.

The enzyme solution after heat precipitation (1 ml) was applied to a MonoQ HR 5/5 anion-exchange column (GE Healthcare) equilibrated with buffer C. The column was washed with 5 ml of buffer C and developed with a 30-ml linear gradient of 0 to 0.3 M KCl in buffer C at a flow rate of 0.5 ml min−1. Succinic semialdehyde reductase eluted between 100 and 180 mM KCl, with the maximum around 150 mM.

(c) Gel filtration chromatography.

The concentrated fraction obtained from the MonoQ-Sepharose chromatography step was applied to a Superdex 200 10/300 GL gel filtration column (10 by 300 mm; volume, 24 ml; GE Healthcare) that had been equilibrated with 20 mM Tris-HCl (pH 7.8) containing 100 mM NaCl. The flow rate was 0.5 ml min. The active protein eluted with a retention volume of 12.5 to 16.5 ml, with the maximum at 14 ml.

(v) Succinyl-CoA reductase.

The enzyme was prepared from 5 g (wet mass) of autotrophically grown M. sedula cells.

(a) DEAE-Sepharose chromatography.

Cell extract (5 ml) was applied to a DEAE-Sepharose column (fast flow; diameter, 1.6 cm; volume, 5 ml; GE Healthcare) that had been equilibrated with buffer C at a flow rate of 2 ml min−1. The activity eluted in the flowthrough. The most active fraction (3 ml) was kept frozen at −20°C in 30% glycerol until it was used.

(b) MonoQ-Sepharose chromatography.

The active fraction obtained from the DEAE-Sepharose chromatography step was applied to a MonoQ HR 5/5 anion-exchange column (GE Healthcare) equilibrated with buffer C. The column was washed with 7 ml of buffer C and developed with an 8-ml linear gradient of 0 to 100 mM KCl in buffer C at a flow rate of 0.5 ml min−1. The activity eluted between 45 and 100 mM KCl, with the maximum around 80 mM. The most active fractions were pooled (2 ml) and kept frozen at −20°C until they were used.

(c) Carboxymethylcellulose chromatography.

The MonoQ-Sepharose fraction was concentrated to a volume of 1 ml using a 10-kDa Amicon Ultra-15 centrifugal filter device (Millipore) and then washed with 5 ml 10 mM Tris-MES (pH 6.5) (designated buffer D). This fraction was then applied to a 1-ml carboxymethylcellulose (CM52) column (Whatman) equilibrated with buffer D. The column was washed with 12 ml buffer D and developed with a 30-ml linear gradient of 0 to 1 M KCl in buffer D at a flow rate of 0.5 ml min−1. The activity eluted between 40 mM and 80 mM salt. Active fractions were kept frozen at −20°C.

(vi) Recombinant Msed_1774 and Msed_1119.

Frozen E. coli cells (0.3 g) were suspended in 0.3 ml of 10 mM Tris-HCl (pH 7.8) containing 50 μg DNase I ml−1. Glass beads (1.2 g; diameter, 0.1 to 0.25 mm) were added, and the suspension was treated for 10 min at 30 Hz and 4°C with a mixer mill (type MM2; Retsch, Haare, Germany). After centrifugation (15 min, 20,000 × g, 4°C), the supernatant (0.3 ml) was incubated at 75°C for 15 min and then cooled on ice for 15 min, which was followed by centrifugation at 20,000 × g and 4°C for 15 min.

Heterologous expression of M. sedula genes in E. coli.

Genes were amplified from M. sedula chromosomal DNA by performing PCR with Pfu polymerase. The sequence of the cloned inserts was determined. Competent E. coli Rosetta 2(DE3) cells that carry a plasmid with genes for rare tRNA species were transformed with the plasmid, grown in 1- or 10-liter fermentors at 37°C in lysogeny broth containing 100 μg of ampicillin ml−1 and 34 μg of chloramphenicol ml−1, and induced at an optical density of 0.7 with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After additional growth for 3 h at 37°C, the cells (approximately 3 g [wet mass] per liter) were harvested and stored at −20°C until they were used.

(i) Malonic semialdehyde reductase.

The malonic semialdehyde reductase gene was amplified by using forward primer (5′-CAATATAAGCACATATGACTGAAAAGGTATC-3′) introducing an NdeI site (underlined) at the initiation codon and reverse primer (5′-CAGAGGCCATCCTCTTCAAGCTTACGGACTC −3′) introducing a HindIII site (underlined) after the stop codon. The PCR conditions were as follows: 35 cycles of denaturation for 45 s at 94°C, primer annealing for 45 s at 60°C, and elongation for 2 min at 72°C. The PCR product was isolated and cloned into pUC18, resulting in plasmid pUC18Msed_1993. Ligation of the NdeI/HindIII fragment of pUC18Msed_1993 into similarly restricted pT7-7 (43) resulted in plasmid pT7-7Msed_1993.

(ii) Succinic semialdehyde reductase.

The succinic semialdehyde reductase gene was amplified using forward primer (5′-CTTTCATGAATGAAAGCTGCAGTACTTC-3′) introducing a BspHI site (underlined) at the initiation codon and reverse primer (5′-CCTCGGGATCCGCTTACGGGATTATG-3′) introducing a BamHI site (underlined) after the stop codon. The PCR conditions were as follows: 30 cycles of denaturation for 45 s at 94°C, primer annealing for 45 s at 55°C, and elongation for 140 s at 72°C. The PCR product was isolated. Ligation of the BspHI/BamHI fragment into NcoI/BamHI-restricted pTrc99a (5) resulted in plasmid pTrc99aMsed_1424.

(iii) Msed_1774.

The Msed_1774 gene was amplified using forward primer (5′-GGCCCATATGATTCCAATTATTCTAGGCGG-3′) introducing an NdeI site (underlined) at the initiation codon and reverse primer (5′-GGGTTTCACTATCAAGCTTCAGGCC −3′) introducing a HindIII site (underlined) after the stop codon. The PCR conditions were as follows: 35 cycles of denaturation for 45 s at 94°C, primer annealing for 45 s at 60°C, and elongation for 3 min at 72°C. The PCR products were isolated and cloned into pUC18, resulting in plasmid pUC18Msed_1774. Ligation of the NdeI/HindIII fragment of pUC18Msed_1774 into similarly restricted pT7-7 (43) resulted in plasmid pT7-7Msed_1774.

(iv) Msed_1119.

The Msed_1119 gene was amplified using forward primer (5′-GGGTACATATGGAAGCTCTAATAGGAGG-3′) introducing an NdeI site (underlined) at the initiation codon and reverse primer (5′-GCGCGCTGGAAAGCTTATGGAAATAG-3′) introducing a HindIII site (underlined) after the stop codon. The PCR conditions were as follows: 35 cycles of denaturation for 45 s at 94°C, primer annealing for 45 s at 55°C, and elongation for 3 min at 72°C. The PCR products were isolated and cloned into pUC18, resulting in plasmid pUC18Msed_1119. Ligation of the NdeI/HindIII fragment of pUC18Msed_1119 into similarly restricted pT7-7 (43) resulted in plasmid pT7-7Msed_1119.

Protein analysis methods.

Protein contents were determined by the method of Bradford (10), using bovine serum albumin as the standard. Protein fractions were analyzed by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (SDS-PAGE) (33). Proteins were visualized by Coomassie brilliant blue R-250 staining (50). In-gel protein digestion with trypsin and matrix-assisted laser desorption ionization mass spectrometry analysis of peptides were carried out by TopLab (Martinsried, Germany). Proteins were identified using ProFound (Proteometrics) software and the NCBI database. Purified recombinant succinic semialdehyde reductase (2.0 mg) was analyzed for metal content by inductively coupled plasma emission spectroscopy at the Chemical Analysis Laboratory of R. Auxier (University of Georgia, Athens). SDS-PAGE (12.5%) was performed as described previously (33). The native molecular mass was determined using a 24-ml Superdex 200 10/300 GL gel filtration column (GE Healthcare) calibrated with vitamin B12 (1.35 kDa), RNase A (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 and 134 kDa), aldolase (158 kDa), catalase (240 kDa), ferritin (440 kDa), and blue dextran 2000 (2000 kDa).

Computational analysis.

BLAST searches were performed using the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/) (4, 8). The amino acid sequences were aligned using CLUSTALW (45) implemented in BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic trees were reconstructed using neighbor-joining algorithms (38) in the TREECONW program package (47).

RESULTS

Malonic semialdehyde reductase activity in cell extracts of M. sedula.

In a coupled spectrophotometric enzyme assay performed at 65°C (the optimal temperature for growth is 75°C) the substrate malonic semialdehyde was enzymatically synthesized by using excess purified recombinant malonyl-CoA reductase (1). This enzyme catalyzes the NADPH-dependent reduction of malonyl-CoA to malonic semialdehyde. When malonic semialdehyde synthesis was complete, M. sedula cell extract was added to measure the further NADPH-dependent reduction of malonic semialdehyde to 3-hydroxypropionate. The specific activity of this conversion in autotrophically grown cells was 1.8 μmol min−1 mg−1 protein, whereas in heterotrophically grown cells the specific activity was 0.35 μmol min−1 mg−1 protein. As a control, malate dehydrogenase activity was measured. The specific activity of this enzyme in autotrophically grown cells was 1.9 μmol min−1 mg−1, compared with 3.5 μmol min−1 mg−1 in heterotrophically grown cells.

Partial purification of malonic semialdehyde reductase from M. sedula.

The enzyme that catalyzes malonic semialdehyde reduction to 3-hydroxypropionate was partially purified from an extract of autotrophically grown cells. The level of enrichment after four purification steps was 145-fold, and the yield was approximately 44% (Table (Table1).1). SDS-PAGE analysis revealed a prominent 33-kDa band (Fig. (Fig.2A).2A). This band was analyzed by in-gel digestion with trypsin and peptide fingerprint mass spectrometry, followed by comparison with the genome of M. sedula (6). The best hit was with the gene gi|146304741 (100% identity, 54% coverage), annotated as 3-hydroxyacyl-CoA dehydrogenase (NAD binding) gene, encoding a 35.3-kDa protein (Msed_1993; 314 amino acids).

FIG. 2.
SDS-PAGE (12.5%) of fractions obtained during purification of native and recombinant malonic semialdehyde reductase from M. sedula. Proteins were stained with Coomassie blue. (A) Enzyme fractions obtained during purification of the native enzyme. ...
TABLE 1.
Partial purification of malonic semialdehyde reductase from M. sedula and purification of heterologously expressed malonic semialdehyde reductase (Msed_1993)

Heterologous expression of malonic semialdehyde reductase from M. sedula in E. coli and characterization of the recombinant enzyme.

The malonic semialdehyde reductase gene encoding the Msed_1993 protein was cloned into the expression vector pT7-7 (43) and expressed in E. coli Rosetta 2(DE3). The overproduced enzyme was soluble and could be purified with one purification step after heat precipitation (Table (Table11 and Fig. Fig.2B).2B). The preparation obtained was virtually pure and produced a single 33-kDa band (expected value, 35.3 kDa) in an SDS-PAGE gel (Fig. (Fig.2B).2B). The purified enzyme catalyzed the expected reaction at a rate of 200 μmol min−1 mg−1 protein at 65°C. The UV-visible spectrum had an absorption maximum around 280 nm. The absorption coefficient at 280 nm was 58,000 M−1 cm−1. The native molecular mass determined by gel filtration was 105 ± 10 kDa, suggesting a homotrimeric subunit composition. The optimum pH (adjusted at 20°C) was 7.2 (pH at 65°C, 6.7), and there was half-maximal activity at pH 9.3 (adjusted at 20°C). The Vmax, 200 μmol min−1 mg−1, corresponds to a turnover value of 115 s−1. The apparent Km for malonic semialdehyde was 70 ± 10 μM, and the apparent Km for NADPH was 70 ± 20 μM. NADH (20% activity) could partially substitute for NADPH (100%). Succinic semialdehyde, acetaldehyde, butyraldehyde, propionaldehyde, or glutaraldehyde did not serve as a substrate (Table (Table2).2). The aldehydes tested did not inactivate the enzyme, as shown by starting the reaction with malonic semialdehyde by adding malonyl-CoA and malonyl-CoA reductase, which resulted in an overall stoichiometry of 2 NADPH molecules oxidized per malonyl-CoA molecule added. Incubation with 10 mM EDTA for 16 h did not inactivate the enzyme, nor did addition of 10 mM EDTA to the assay mixture inhibit enzyme activity. Addition of Zn2+ (0 to 40 μM) or Mg2+ or Mn2+ (0 to 5 mM) to the enzyme assay mixture neither stimulated nor inactivated the enzyme.

TABLE 2.
Molecular and catalytic properties of succinic semialdehyde reductase, malonic semialdehyde reductase, and malonyl-CoA/succinyl-CoA reductase

Succinic semialdehyde reductase activity in cell extracts of M. sedula.

Extracts were tested to examine the succinic semialdehyde-dependent oxidation of NAD(P)H at 65°C. Extracts of autotrophically grown cells catalyzed the NADPH-dependent reaction with a specific activity of 1.4 μmol NADPH oxidized min−1 mg−1 of protein. When measured with the same concentration of NADH (0.5 mM), the specific activity was 0.15 μmol min−1 mg−1 of protein. No (<0.01 μmol min−1 mg−1 of protein) succinic semialdehyde-dependent oxidation of NADPH or NADH was observed with heterotrophically grown cells.

Partial purification of succinic semialdehyde reductase from M. sedula.

The enzyme that catalyzes reduction of succinic semialdehyde to 4-hydroxybutyrate was partially purified from an extract of autotrophically grown cells. The level of enrichment after three purification steps was 420-fold, and the yield was approximately 7% (Table (Table3).3). In an SDS-PAGE gel a prominent band was detected at 42 kDa (Fig. (Fig.3A).3A). This band was analyzed by in-gel digestion with trypsin and peptide fingerprint mass spectrometry, followed by comparison with the genome of M. sedula (6). The best hit was with the gene gi|146304190 (100% identity, 65% coverage) annotated as a Zn-dependent alcohol dehydrogenase gene encoding a 37.9-kDa protein (Msed_1424; 360 amino acids).

FIG. 3.
SDS-PAGE (12.5%) of fractions obtained during purification of native and recombinant succinic semialdehyde reductase from M. sedula. Proteins were stained with Coomassie blue. (A) Enzyme fractions obtained during purification of the native enzyme. ...
TABLE 3.
Partial purification of succinic semialdehyde reductase from M. sedula and purification of heterologously expressed succinic semialdehyde reductase (Msed_1424)

Heterologous expression of succinic semialdehyde reductase from M. sedula in E. coli and characterization of the recombinant enzyme.

The succinic semialdehyde reductase gene Msed_1424 was expressed in E. coli Rosetta 2(DE3). The overproduced enzyme was purified using four purification steps (Table (Table33 and Fig. Fig.3B).3B). The preparation obtained was virtually pure and produced a single band at 40 kDa (expected value, 37.9 kDa) on an SDS-PAGE gel (Fig. (Fig.3B).3B). The purified enzyme catalyzed the expected reaction at a rate of 700 μmol min−1 mg−1 protein at 65°C. The UV-visible spectrum had an absorption maximum at around 280 nm. The absorption coefficient at 280 nm was 15,000 M−1 cm−1. The native molecular mass determined by gel filtration was 74 ± 7 kDa, suggesting a homodimeric subunit composition. The optimum pH at 65°C was 7.5 (pH at 20°C, 7.9), and there were half-maximal activities at pH values at 65°C of 6.5 and 8 (pH at 20°C, 6.9 and 8.4). The Vmax at 65°C (700 μmol min−1 mg−1) corresponded to a turnover value of 440 s−1, the apparent Km for succinic semialdehyde (determined with NADPH) was 52 ± 8 μM, the apparent Km for NADPH was 6 ± 2 μM, and the apparent Km for NADH was 180 ± 25 μM. Malonic semialdehyde, acetaldehyde, butyraldehyde, propionaldehyde, or glutaraldehyde did not serve as a substrate, nor did these compounds inactivate the enzyme (Table (Table2).2). Incubation with 10 mM EDTA for 16 h or with 100 mM EDTA for 1 h at temperatures up to 65°C did not inactivate the enzyme, nor did addition of 10 mM EDTA to the assay mixture inhibit enzyme activity. Addition of Zn2+ (0 to 40 μM), Mg2+ or Mn2+ (0 to 5 mM), or Fe2+ (0 to 1 mM) to the enzyme assay mixture did not stimulate or inactivate the enzyme. Metal analysis (27 elements) of succinic semialdehyde reductase using plasma emission spectroscopy revealed the presence of 1.9 mol of zinc per mol of enzyme monomer, whereas other metals could not be detected.

Succinyl-CoA reductase activity in cell extracts of M. sedula.

Extracts were tested for succinyl-CoA-dependent oxidation of NAD(P)H. The rates were calculated based on the assumption that the enzyme catalyzed only succinyl-CoA reduction to succinic semialdehyde. Further reduction to 4-hydroxybutyrate by succinic semialdehyde reductase causes the oxidation of another NADPH molecule. Extracts of autotrophically grown cells exhibited an NADPH-dependent succinyl-CoA reductase activity of 0.2 μmol min−1 mg−1 of soluble protein, whereas extracts of heterotrophically grown cells exhibited an activity of 10 nmol min−1 mg−1 of protein. No activity was observed with NADH. When malonyl-CoA was added instead of succinyl-CoA, the same activity was measured.

Partial purification and characterization of succinyl-CoA reductase from M. sedula.

The enzyme catalyzing reduction of succinyl-CoA to succinic semialdehyde was partially purified from an extract of autotrophically grown cells. The level of enrichment after four purification steps was 37-fold, and the yield was approximately 2% (Table (Table4).4). The enzyme preparation catalyzed malonyl-CoA and succinyl-CoA reduction with NADPH at the same rate (7.5 μmol min−1 mg−1 protein). In an SDS-PAGE gel a prominent band was detected at 43 kDa (Fig. (Fig.4).4). This band was analyzed by in-gel digestion with trypsin and peptide fingerprint mass spectrometry, followed by comparison with the genome of M. sedula (6). The only hit was with the gene gi|146303492 (100% identity, 54% coverage) annotated as an aspartate-semialdehyde dehydrogenase gene coding for a 39.2-kDa protein (Msed_0709; 357 amino acids). This protein was identified previously as malonyl-CoA reductase (1). The pure recombinant malonyl-CoA reductase from the closely related organism S. tokodaii was also examined. This enzyme showed succinyl-CoA reductase specific activity at 65°C of 40 μmol min−1 mg−1 protein, corresponding to a turnover value of 26 s−1. The specific malonyl-CoA reduction rate of the Sulfolobus enzyme (1) was 44 μmol min−1 mg−1 protein. The apparent Km for succinyl-CoA was 150 ± 15 μM, and the apparent Km for NADPH was 190 ± 25 (Table (Table2).2). These data showed that malonyl-CoA reductase is a promiscuous enzyme and acts nearly equally well with malonyl-CoA and succinyl-CoA.

FIG. 4.
SDS-PAGE (12.5%) of fractions obtained during purification of native succinyl-CoA reductase from M. sedula. Proteins were stained with Coomassie blue. Lane 1, cell extract of autotrophically grown cells (20 μg); lane 2, enzyme fraction ...
TABLE 4.
Partial purification of succinyl-CoA reductase (Msed_0709) from M. sedula

Investigation of reductases from M. sedula that show high levels of similarity to succinyl-CoA reductase from T. neutrophilus.

The succinyl-CoA reductase from T. neutrophilus (Tneu_0421) is not homologous to the enzyme from M. sedula (37). However, the M. sedula genome harbors two genes that are very similar to the succinyl-CoA reductase gene from T. neutrophilus, namely Msed_1774 (50% amino acid sequence identity and 72% similarity) and Msed_1119 (43% amino acid sequence identity and 65% similarity). Both genes were cloned and expressed in E. coli Rosetta 2(DE3). The overproduced enzymes were soluble and were partially purified by heat precipitation. These enzymes did not catalyze NAD(P)H-dependent reduction of succinyl-CoA. Yet the approximately 80% pure enzymes showed succinic semialdehyde dehydrogenase activity with NADP+ (Msed_1774, 10 μmol min−1 mg−1 protein; Msed_1119, 0.5 μmol min−1 mg−1 protein), whereas NAD+ was not reduced. Since CoA was not required and did not stimulate the reaction, these aldehyde dehydrogenases are not CoA-acylating enzymes and therefore form succinate rather than succinyl-CoA. Acetaldehyde, butyraldehyde, propionaldehyde, or glutaraldehyde did not serve as a substrate. Hence, the putative succinyl-CoA reductase genes described above do not code for succinyl-CoA reductase.

DISCUSSION

Function and properties of the enzymes.

We purified and characterized three oxidoreductases from autotrophically grown cells of M. sedula that function in the autotrophic CO2 fixation cycle (Fig. (Fig.1)1) (9). The strong upregulation of these enzyme activities under autotrophic conditions supports their ascribed functions as reductases rather than dehydrogenases. Also, the NADPH specificity suggests that they function as reductases in an anabolic pathway rather than as dehydrogenases in a catabolic pathway. The specific activities are high enough to explain autotrophic growth with a generation time of 20 h. The apparent Km values for their substrates and cosubstrates are rather low and are in line with the proposed enzyme functions. The properties of the enzymes are summarized in Table Table22.

Malonic semialdehyde reductase (Fig. (Fig.1)1) was NADPH dependent; the activity with NADH was fivefold lower than the activity with NADPH, and the apparent Km with NADH was much higher than the apparent Km with NADPH. Surprisingly, succinic semialdehyde was not used as a substrate. This enzyme, annotated as a putative 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), exhibits a Rossmann fold NAD(P)H/NAD(P)+ binding domain at the N terminus (amino acids 17 to 182) with the characteristic consensus binding pattern GAGVIG starting at amino acid 9. The C terminus has a 3-hydroxyacyl-CoA dehydrogenase domain. This enzyme cannot be classified unambiguously, and no divalent metal ion was required.

Succinic semialdehyde reductase (EC 1.1.1.2) (Fig. (Fig.1)1) strongly prefers NADPH as the electron donor and does not act on malonic semialdehyde. It was annotated as a Zn-dependent class III alcohol dehydrogenase with an N-terminal GroES-like domain (amino acids 25 to 151) that is also found in other medium-chain alcohol dehydrogenases (36) and a Rossmann fold NAD(P)H/NAD(P)+ binding domain (amino acids 181 to 321) with the characteristic consensus binding pattern GVGGVG starting at amino acid 189. Dependence on Zn2+ and various other divalent metal ions could not be demonstrated, nor did EDTA inhibit or inactivate the enzyme. Comparison with the well-studied horse liver alcohol dehydrogenase showed the presence of the strictly conserved sequence motif GHE of the catalytic zinc binding site (amino acids 58 to 60) with two flanking cysteines (amino acids 38 and 165). A conserved sequence for a structural zinc binding site, CXCXXCXXXXXXXC (cysteines at amino acids 89, 92, 95 and 103), was found (7, 46). Apparently, both zinc binding sites are loaded with zinc since the enzyme contains 1.9 mol of zinc per mol of enzyme monomer. Zinc is not accessible by EDTA even at a high temperature, possibly due to the high thermal stability of the reductase (34). The succinic semialdehyde reductase of M. sedula is only distantly related to 4-hydroxybutyrate dehydrogenases of Clostridium kluyveri (48) and Arabidopsis thaliana (11).

Succinyl-CoA/malonyl-CoA reductase is a promiscuous enzyme, which is in remarkable contrast to the previously studied substrate-specific semialdehyde reductases. This enzyme is the second promiscuous enzyme in this cycle; the other is acetyl-CoA/propionyl-CoA carboxylase (12, 29). Succinyl-CoA/malonyl-CoA reductase catalyzes not only the reduction of succinyl-CoA (apparent Km, 0.15 mM; Vmax, 40 U mg−1) but also the reduction of malonyl-CoA (apparent Km, 0.04 mM; Vmax, 44 U mg−1) (Table (Table2).2). In our previous study of malonyl-CoA reductase (1), the ability to reduce succinyl-CoA efficiently was erroneously overlooked. An unrelated succinyl-CoA reductase is part of the autotrophic dicarboxylate/4-hydroxybutyrate cycle in T. neutrophilus (37). The reductases in M. sedula encoded by homologous genes (Msed_1774 and Msed_1119) do not function as succinyl-CoA reductases. Rather, they show NADP+-dependent succinic semialdehyde dehydrogenase activity (non-CoA acylating). Similar enzymes have been reported for Sulfolobus solfataricus (17). The natural aldehyde substrate and function of the Msed_1119 protein are at issue since the specific activity with succinic semialdehyde is rather low (0.5 μmol min−1 mg−1 protein).

Localization of genes in the M. sedula genome and distribution of the genes.

The succinic semialdehyde reductase gene is located two genes downstream of the acryloyl-CoA reductase gene (Msed_1426), and the malonic semialdehyde reductase gene is located eight genes downstream of the 3-hydroxypropionyl-CoA dehydratase gene (Msed_2001) (44). The gene for malonyl-CoA/succinyl-CoA reductase (Msed_0709) is located between genes encoding hypothetical proteins; no known gene of the 3-hydroxypropionate/4-hydroxybutyrate cycle is located nearby.

The conversion of acetyl-CoA via 3-hydroxypropionate to succinyl-CoA, as studied here in a member of the Sulfolobales, is also part of the autotrophic 3-hydroxypropionate cycle in Chloroflexus. Obviously, the two autotrophic pathways have evolved independently in the two lineages since they involve completely different genes coding for malonyl-CoA reductase (30), malonic semialdehyde reductase (this study), 3-hydroxypropionyl-CoA synthetase (2, 3), 3-hydroxypropionyl-CoA dehydratase (44), and acryloyl-CoA reductase (44). Likewise, the conversion of succinyl-CoA via 4-hydroxybutyrate to acetyl-CoA is also part of the autotrophic dicarboxylate/4-hydroxybutyrate cycle in Desulfurococcales (27) and Thermoproteales (37).

Similar succinic semialdehyde reductase genes are present in autotrophic members of the Sulfolobales (Metallosphaera sp., Sulfolobus spp.) and Thermoproteales, and they probably have the same function that they have in M. sedula (Fig. (Fig.5).5). The homologous genes in Nitrosopumilus maritimus (gi|161528031; 30% identity and 48% similarity) and Cenarchaeum symbiosum (gi|118576590; 31% identity and 45% similarity), which are members of the mesophilic marine group I Crenarchaea, are only distantly related to the Sulfolobales genes, and at this point it is not possible to deduce their function.

FIG. 5.
Phylogenetic tree of succinic semialdehyde reductase (ssr) homologues from Archaea (expectation value in a standard NCBI BLASTP search, <e−90; C. symbiosum A and N. maritimus were searched separately). The homologues of the M. sedula enzyme ...

Malonic semialdehyde reductase genes are also present in the genomes of other autotrophic members of the Sulfolobales (Fig. (Fig.6).6). As expected, such genes are not found in the genomes of members of the Thermoproteales and Desulfurococcales. The functions of the less similar genes in N. maritimus (gi|161528031; 39% identity and 63% similarity) and C. symbiosum (gi|118575609; 38% identity and 59% similarity), as well as in Archeaoglobus fulgidus (gi|11498805; 42% identity and 64% similarity) and Planctomyces maris (gi|161528031; 37% identity and 59% similarity), cannot be predicted. These genes may encode malonic semialdehyde reductase, as they do in M. sedula, or, e.g., 3-hydroxyacyl-CoA dehydrogenase. The capacity to oxidize 3-hydroxypropionate to malonic semialdehyde or to reduce malonic semialdehyde to 3-hydroxypropionate occurs in all kingdoms of organisms (14).

FIG. 6.
Phylogenetic tree of malonic semialdehyde reductase homologs from Archaea (expectation value in a standard NCBI BLASTP search, <e−57). The homologues of the M. sedula enzyme gene in Sulfolobales most likely encode malonic semialdehyde ...

The distribution of malonyl-CoA/succinyl-CoA reductase, a paralogue of aspartate semialdehyde dehydrogenase, has been discussed previously (1, 9). A phylogenetic tree of the gene (1, 9) suggests that there was duplication of the aspartate semialdehyde dehydrogenase gene in the ancestor of the Sulfolobales and that there was independent evolution of the two genes to perform different functions. This does not apply to C. symbiosum and N. maritimus, which have only one copy of the aspartate semialdehyde dehydrogenase gene, leaving the question, what kind of malonyl-CoA reductase do these organisms use if they use a 3-hydroxypropionate/4-hydroxybutyrate cycle for CO2 fixation.

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft.

We thank Christa Ebenau-Jehle and Nasser Gad'on for technical assistance.

Footnotes

[down-pointing small open triangle]Published ahead of print on 14 August 2009.

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