Identifying the missing steps of the autotrophic 3-hydroxypropionate CO2 fixation cycle in Chloroflexus aurantiacus

Abstract

The phototrophic bacterium Chloroflexus aurantiacus uses a yet unsolved 3-hydroxypropionate cycle for autotrophic CO(2) fixation. It starts from acetyl-CoA, with acetyl-CoA and propionyl-CoA carboxylases acting as carboxylating enzymes. In a first cycle, (S)-malyl-CoA is formed from acetyl-CoA and 2 molecules of bicarbonate. (S)-Malyl-CoA cleavage releases the CO(2) fixation product glyoxylate and regenerates the starting molecule acetyl-CoA. Here we complete the missing steps devoted to glyoxylate assimilation. In a second cycle, glyoxylate is combined with propionyl-CoA, an intermediate of the first cycle, to form beta-methylmalyl-CoA. This condensation is followed by dehydration to mesaconyl-C1-CoA. An unprecedented CoA transferase catalyzes the intramolecular transfer of the CoA moiety to the C4 carboxyl group of mesaconate. Mesaconyl-C4-CoA then is hydrated by an enoyl-CoA hydratase to (S)-citramalyl-CoA. (S)-Citramalyl-CoA is cleaved into acetyl-CoA and pyruvate by a tri-functional lyase, which previously cleaved (S)-malyl-CoA and formed beta-methylmalyl-CoA. Thus, the enigmatic disproportionation of glyoxylate and propionyl-CoA into acetyl-CoA and pyruvate is solved in an elegant and economic way requiring only 3 additional enzymes. The whole bicyclic pathway results in pyruvate formation from 3 molecules of bicarbonate and involves 19 steps but only 13 enzymes. Elements of the 3-hydroxypropionate cycle may be used for the assimilation of small organic molecules. The 3-hydroxypropionate cycle is compared with the Calvin-Benson-Bassham cycle and other autotrophic pathways.

Fig. 1.

The complete 3-hydroxypropionate cycle, as studied in C. aurantiacus. [1] Acetyl-CoA carboxylase, [2] malonyl-CoA reductase, [3] propionyl-CoA synthase, [4] propionyl-CoA carboxylase, [5] methylmalonyl-CoA epimerase, [6] methylmalonyl-CoA mutase, [7] succinyl-CoA:(S)-malate-CoA transferase, [8] succinate dehydrogenase, [9] fumarate hydratase, [10 a, b, c] (S)-malyl-CoA/β-methylmalyl-CoA/(S)-citramalyl-CoA (MMC) lyase, [11] mesaconyl-C1-CoA hydratase (β-methylmalyl-CoA dehydratase), [12] mesaconyl-CoA C1-C4 CoA transferase, [13] mesaconyl-C4-CoA hydratase. Carbon-labeling patterns during the interconversion of propionyl-CoA plus glyoxylate to pyruvate plus acetyl-CoA via C₅ compounds are shown. ¹⁴C carbon atoms derived from [1-¹⁴C]propionyl-CoA are marked by ▴, and ¹³C carbon atoms derived from [1,2,3-¹³C]propionyl-CoA are marked by ■. Note that the cleavage of citramalyl-CoA requires that the CoA moiety be shifted finally from the “right” carboxyl group of β-methylmalyl-CoA to the “left” carboxyl group of citramalyl-CoA. This shifting is accomplished by an intramolecular CoA transfer (reaction 12). Otherwise, citramalyl-CoA cleavage into pyruvate and acetyl-CoA would not be feasible.

Fig. 2.

Characterization of substrates and products of the conversion of mesaconyl-C1-CoA to mesaconyl-C4-CoA. (A) UV spectra of propionyl-CoA, β-methylmalyl-CoA, mesaconyl-C1-CoA, and mesaconyl-C4-CoA. (B) HPLC separation of 2 chemically synthesized isomeric forms of mesaconyl-CoA carrying the CoA moiety randomly at either C1 or C4 (Bottom) compared with the separation of enzymatically formed mesaconyl-C1-CoA and its product (Upper). A 40-mL gradient from 2% to 10% acetonitrile in 40 mM K₂HPO₄/HCOOH buffer (pH 4.2), with a flow rate of 1 mL min⁻¹, was used with a reversed-phase column. Detection was at 260 nm. (C) TLC separation of labeled products after alkaline hydrolysis of the CoA thioester products formed from [1-¹⁴C]propionyl-CoA and glyoxylate. Detection was by phosphoimaging. Lane 1, mesaconyl-C1-CoA; lane 2, observed product. S, start; M, mesaconate. (D) Spectrophotometric assay used for the characterization of the mesaconyl-CoA C1-C4 CoA transferase. Mesaconyl-C4-CoA hydratase, MMC lyase, and lactate dehydrogenase were used as coupling enzymes. The reaction could be started by either mesaconyl-C1-CoA or mesaconyl-CoA C1-C4 CoA transferase. In the presence of mesaconyl-CoA C1-C4 CoA transferase mesaconyl-C1-CoA (here 0.2 mM) is converted to mesaconyl-C4-CoA, which is hydrated to (S)-citramalyl-CoA by mesaconyl-C4-CoA hydratase. (S)-Citramalyl-CoA subsequently is cleaved into acetyl-CoA and pyruvate by MMC lyase. Pyruvate then is reduced by lactate dehydrogenase to lactate under NADH consumption, followed spectrophotometrically at 365 nm.

Fig. 3.

Organization of genes involved in the glyoxylate assimilation cycle. For catalyzed reactions (in parentheses) see Fig. 1. To amplify the intergenic regions of the cluster shown below, standard PCRs were performed with cDNA (a) and genomic DNA (b) as control. The positions of the amplified fragments are indicated by bars, and their expected sizes are given in bp. Lane M contained a 100-bp DNA ladder. meh, mesaconyl-C4-CoA hydratase (mesaconyl-C4-CoA (enoyl-CoA) hydratase) (reaction 13); smtAB, succinyl-CoA:(S)-malate CoA transferase subunits A and B (reaction 7); mct, mesaconyl-CoA C1-C4 CoA transferase (reaction 12); mcl, trifunctional (S)-malyl-CoA/β-methylmalyl-CoA/(S)-citramalyl-CoA (MMC) lyase (reactions 10 a, b, c); mch, mesaconyl-C1-CoA hydratase (β-methylmalyl-CoA dehydratase) (reaction 11). Between the genes of smtB and mct there are 2 ORFs of unknown function (no similar proteins are found in the database). For primers, see Table S1.

Fig. 4.

HPLC separation of ¹⁴C-labeled products formed from [1-¹⁴C]propionyl-CoA and glyoxylate by different purified recombinant enzymes. (A) Before addition of enzyme. (B) After the addition of MMC lyase. (C) As B, plus mesaconyl-C1-CoA hydratase. (D) As C, plus mesaconyl-CoA C1-C4 CoA transferase. (E) As D, plus mesaconyl-C4-CoA hydratase. (F) As E: formation of non-labeled acetyl-CoA and ¹⁴C-pyruvate after additional incubation time. A reversed-phase column was developed for 7 min under isocratic conditions with 100 mM NaH₂PO₄ (pH 4.0) in 7.5% methanol (vol/vol), followed by a linear 10-min gradient from 0% to 60% of 100 mM sodium acetate (pH 4.6) in 90% methanol (vol/vol) at a flow rate of 1 mL min⁻¹. Acetyl-CoA was detected by diode array detection because of its absorption at 260 nm. Radioactivity was detected by solid-state scintillation counting.