Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

Abstract

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.

FIG. 1.

Part of the bicyclic autotrophic CO₂ assimilation pathway in C. aurantiacus. The scheme shows the last two reactions of the first cycle and the known reactions of the second cycle. In the first cycle (3-hydroxypropionate cycle), two HCO₃⁻ are fixed to form glyoxylate as the first net fixation product. In the second cycle (glyoxylate assimilation cycle), glyoxylate and propionyl-CoA are condensed to erythro-β-methylmalyl-CoA (probably [2R,3S]-2-methylmalyl-CoA), which is converted to acetyl-CoA and pyruvate. SmtAB, succinyl-CoA:l-malate CoA transferase; Mcl, l-malyl-CoA lyase/erythro-β-methylmalyl-CoA lyase; Mcd, β-methylmalyl-CoA dehydratase; Sct, succinyl-CoA:R-citramalate CoA transferase; Ccl, R-citramalyl-CoA lyase.

FIG. 2.

Results of denaturing PAGE (12.5%) of enzyme fractions obtained during the purification of recombinant R-citramalyl-CoA lyase of C. aurantiacus from E. coli (10 μg each). Lanes: 1, cells before induction; 2, cells after induction; 3, cell extract after heat precipitation (10 min at 65°C, followed by 10 min at 75°C); 4, sample after ammonium sulfate precipitation; 5, size exclusion fraction; 6, MonoQ fraction; 7, molecular mass markers. The arrows indicate the molecular mass markers (rabbit phosphorylase b, 97 kDa; bovine serum albumin, 67 kDa; egg ovalbumin, 45 kDa; lactate dehydrogenase, 34 kDa; carbonic anhydrase, 29 kDa; lysozyme, 14 kDa). The gel was stained with Coomassie brilliant blue R-250.

FIG. 3.

Important gene clusters for autotrophic growth in Chloroflexus aurantiacus and similar genes or gene clusters in other organisms. Corresponding ORFs are shown in the same color. Putative products or ORFs: red, CoA transferase; medium blue, R-citramalyl-CoA lyase; gray, malonyl-CoA reductase; magenta, propionyl-CoA synthase; dark blue, l-malyl-CoA lyase/β-methylmalyl-CoA lyase; dark green, β-methylmalyl-CoA dehydratase; medium green, acetyl-CoA carboxylase; violet, ORFs located on the gene cluster on contig NZ_AAAH02000037 together with the genes for Sct and Ccl; black, ORFs which show no similarities to known proteins required for CO₂ fixation in C. aurantiacus. sct encodes succinyl-CoA:R-citramalate CoA transferase; ccl encodes R-citramalyl-CoA lyase; mcr encodes malonyl-CoA reductase; pcs encodes propionyl-CoA synthase; smtA encodes succinyl-CoA:l-malate CoA transferase, subunit A; smtB encodes succinyl-CoA:l-malate CoA transferase, subunit B (note that the smtAB gene product also has succinyl-CoA:S-citramalate CoA transferase activity); ctr encodes putative CoA transferase; mcl encodes l-malyl-CoA lyase/erythro-β-methylmalyl-CoA lyase (note that the mcl gene product also has S-citramalyl-CoA lyase activity); mcd encodes β-methylmalyl-CoA dehydratase; and accD accA accC encodes subunits of acetyl-CoA carboxylase.

FIG. 4.

Sequence alignment of R-citramalyl-CoA lyase from Chloroflexus aurantiacus to different proteins of the same family. GenBank accession numbers are as follows: for Chloroflexus aurantiacus R-citramalyl-CoA lyase (Ccl), ZP_00768726; for Brucella melitensis 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase), NP_540843; for Homo sapiens HMG-CoA lyase, AAA92733; for Rhodospirillum rubrum HMG-CoA lyase, AAB50182; for Bacillus subtilis HMG-CoA lyase, NP_389705; for Mycobacterium tuberculosis isopropylmalate synthase (LeuA), NP_218227; for Salmonella enterica serovar Typhimurium LeuA, AAL19077; for Leptospira interrogans LeuA, AAS70315; for Methanococcus jannaschii (R)-citramalate synthase (d-citramalate synthase) (CimA), Q58787; and for Azotobacter vinlandii homocitrate synthase (NifV), AAA64727. The numbers in parentheses at the start of some rows in the first sequence alignment block give the number of N-terminal residues in each protein that are omitted from this alignment. Strictly conserved residues are in bold. The circles indicate residues involved in the active site, and the triangle indicates an important residue in HMG-CoA lyases.

FIG. 5.

Similarity tree of homologues of R-citramalyl-CoA lyase (Ccl) from Chloroflexus aurantiacus based on amino acid sequences. The BLOSUM 62 matrix was used for the alignment. The PAM scale indicates the percentage of point-accepted mutations. GenBank accession numbers are as follows: for Methanococcus jannaschii (R)-citramalate synthase (CimA), Q58787; for Salmonella enterica serovar Typhimurium isopropylmalate synthase (LeuA), AAL19077; for Mycobacterium tuberculosis LeuA, NP_218227; for Leptospira interrogans LeuA, AAS70315; for Azotobacter vinelandii homocitrate synthase (NifV), AAA64727; for Bacillus subtilis 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase), NP_389705; for Rhodospirillum rubrum HMG-CoA lyase, AAB50182; for Brucella melitensis HMG-CoA lyase, NP_540843; for Homo sapiens HMG-CoA lyase, AAA92733; and for Chloroflexus aurantiacus R-citramalyl-CoA lyase (Ccl), ZP_00768726.