Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp

Abstract

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.

FIG. 1.

3-Hydroxypropionate cycle of Chloroflexus aurantiacus. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, propionyl-CoA synthase; 4, propionyl-CoA carboxylase; 5, methylmalonyl-CoA epimerase; 6, methylmalonyl-CoA mutase; 7, succinyl-CoA:l-malate-CoA transferase; 8, succinate dehydrogenase; 9, fumarate hydratase; 10, l-malyl-CoA lyase.

FIG. 2.

SDS-PAGE (12.5%) of fractions obtained during purification of native and recombinant malonyl-CoA reductase. Proteins were stained with Coomassie blue. (A) Enzyme fractions during purification of the native enzyme from M. sedula. Lanes: 1, molecular mass standard proteins; 2, cell extract of autotrophically grown cells (20 μg); 3, enzyme fraction after DEAE-Sepharose chromatography (20 μg); 4, enzyme fraction after chromatography on phenyl-Sepharose (5 μg). (B) Heterologous expression of malonyl-CoA reductase gene from S. tokodaii in E. coli Rosetta 2. Lanes: 1, molecular mass standard proteins; 2, cell extract of E. coli before induction (20 μg); 3, cell extract of E. coli after 3 hours of induced growth (20 μg); 4, cell extract of E. coli after heat precipitation (10 μg). (C) Purified recombinant malonyl-CoA reductase from S. tokodaii. Lanes: 1, fraction after gel filtration chromatography and chromatography on Resource-phenyl (10 μg); 2, molecular mass standard proteins.

FIG. 3.

Reactions catalyzed by aspartate-semialdehyde dehydrogenase (ASD) (A) and malonyl-CoA reductase (MCR) (B).

FIG. 4.

(A) Alignment of the N-terminal amino acid sequence of malonyl-CoA reductase (MCR) from M. sedula and two hypothetical proteins from S. tokodaii. S. tokodaii ASD, hypothetical aspartate-semialdehyde dehydrogenase; S. tokodaii MCR, hypothetical malonyl-CoA reductase; M. sedula MCR, N-terminal amino acid sequence of purified malonyl-CoA reductase from M. sedula. The gray boxes indicate the NADPH binding motif (GxxGxxG) and the conserved cysteine and histidine residues. (B) Genetic environment of the hypothetical genes for aspartate-semialdehyde dehydrogenases (ASD) and malonyl-CoA reductase (MCR) from S. acidocaldarius, S. solfataricus, and S. tokodaii; ADH, alcohol dehydrogenase.

FIG. 5.

(A) UV-visible spectrum of purified recombinant malonyl-CoA reductase (0.032 mg/ml). (B) Study of nucleic acid binding to malonyl-CoA reductase. Bound nucleic acid was extracted from protein by the phenol-chloroform extraction method. Samples were separated on a 1.2% agarose gel. Lanes: 1, 50-bp DNA ladder; 2, extracted nucleic acid from malonyl-CoA reductase, without treatment (1 μg); 3, extracted nucleic acid after treatment with RNase (1 μg); 4, extracted nucleic acid after treatment with DNase (1 μg).

FIG. 6.

Phylogenetic tree of homologs of malonyl-CoA reductase (MCR) and aspartate-semialdehyde dehydrogenases (ASD) based on amino acid sequences. The BLOSSUM 62 matrix was used. The PAM scale indicates the percentage of point accepted mutations. V. cholerae, Vibrio cholerae; H. influenzae, Haemophilus influenzae.

FIG. 7.

Proposed reaction mechanism of malonyl-CoA reductase.