Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway

Abstract

Fifty years ago, Kornberg and Krebs established the glyoxylate cycle as the pathway for the synthesis of cell constituents from C2-units. However, since then, many bacteria have been described that do not contain isocitrate lyase, the key enzyme of this pathway. Here, a pathway termed the ethylmalonyl-CoA pathway operating in such organisms is described. Isotopically labeled acetate and bicarbonate were transformed to ethylmalonyl-CoA by cell extracts of acetate-grown, isocitrate lyase-negative Rhodobacter sphaeroides as determined by NMR spectroscopy. Crotonyl-CoA carboxylase/reductase, catalyzing crotonyl-CoA + CO2 + NADPH --> ethylmalonyl-CoA- + NADP+ was identified as the key enzyme of the ethylmalonyl-CoA pathway. The reductive carboxylation of an enoyl-thioester is a unique biochemical reaction, unprecedented in biology. The enzyme from R. sphaeroides was heterologously produced in Escherichia coli and characterized. Crotonyl-CoA carboxylase/reductase (or its gene) can be used as a marker for the presence of the ethylmalonyl-CoA pathway, which functions not only in acetyl-CoA assimilation. In Streptomyces sp., it may also supply precursors (ethylmalonyl-CoA) for antibiotic biosynthesis. For methylotrophic bacteria such as Methylobacterium extorquens, extension of the serine cycle with reactions of the ethylmalonyl-CoA pathway leads to a simplified scheme for isocitrate lyase-independent C1 assimilation.

Fig. 1.

The glyoxylate cycle as proposed by Kornberg and Krebs 50 years ago. The citric acid cycle is modified to bypass the two decarboxylation steps by the action of its two key enzymes isocitrate lyase and malate synthase. This allows the net synthesis of malate from two molecules of acetyl-CoA.

Fig. 2.

HPLC analysis of CoA thioesters formed from acetyl-CoA and NaHCO₃ by cell extracts of R. sphaeroides in the presence of NADPH at 30°C. [U-¹⁴C]acetyl-CoA was synthesized by incubation of 1 mM [U-¹⁴C]acetate (320 kBq ml⁻¹), 4 mM ATP, 1 mM CoA, 5 mM KCl, and 4 mM MgCl₂ with 6 U·ml⁻¹ of acetyl-CoA synthetase in 70 mM Tris·HCl buffer (pH 7.9). (A) The reaction was started by addition of 0.5 mM 5–5′-dithiobis(2-nitrobenzoic acid), 35 mM NaHCO₃, 6.5 mM NADPH, and 0.8 mg of cell-extract protein. (B and C) After 5 min (B) and 45 min (C), incubation samples were withdrawn from the assay mixture and analyzed by reverse-phase HPLC. (D) Products formed in the presence of NaH¹⁴CO₃ (480 kBq·ml⁻¹) after 45 min incubation; unlabeled acetate was used. Products were identified by MS-HPLC, and the elution times were as follows: free CoA, 6.2 min; ethylmalonyl-CoA, 10.6 min; acetyl-CoA, 12.6 min; acetoacetyl-CoA, 14.6 min; crotonyl-CoA, 23 min; and butyryl-CoA, 25 min.

Fig. 3.

Recombinant crotonyl-CoA carboxylase/reductase. (A) Denaturing PAGE of various steps during purification. Lane 1, 25 μg of E. coli cell-extract protein before induction; lane 2, 25 μg of E. coli cell-extract protein after 4 h of induction; lane 3, 20 μg of protein after ultracentrifugation; lane 4, 15 μg of protein from the DEAE column step; lane 5, 8 μg of protein from the Cibacron Blue column step; lane 6, molecular mass marker (97 kDa, phosphorylase B; 67 kDa, BSA; 45 kDa, ovalbumin; 34 kDa, lactate dehydrogenase; 29 kDa, carbonic anhydrase; 14 kDa, lysozyme). (B) Specific activity of the crotonyl-CoA carboxylase/reductase at various steps during purification. The specific activity was determined spectrophotometrically by the crotonyl-CoA-dependent oxidation of NADPH at 360 nm. Numbering is according to A.

Fig. 4.

The ethylmalonyl-CoA pathway as studied in isocitrate lyase-negative R. sphaeroides. Crotonyl-CoA carboxylase/reductase was identified as the key enzyme of the herein-described acetyl-CoA assimilation pathway distinct from the glyoxylate cycle. Mutations in the genes encoding β-ketothiolase and mesaconyl-CoA hydratase were previously shown to result in an acetate-minus phenotype (13). The bifunctional β-methylmalyl-CoA/malyl-CoA lyase catalyses the cleavage of β-methylmalyl-CoA as well as the condensation of acetyl-CoA and glyoxylate to form malyl-CoA (18). Dotted lines indicate steps that have not been elucidated so far. Exogenous CO₂ is not required for growth with acetate as sole carbon source, indicating that the two molecules of CO₂ fixed in the ethylmalonyl-CoA pathway are derived from the oxidation of acetyl-CoA.

Fig. 5.

Proposed pathway for the assimilation of C₁ compounds by isocitrate lyase-negative type II methylotrophs by using the serine cycle for formaldehyde fixation. The ethylmalonyl-CoA pathway described here (excluding the condensation of acetyl-CoA and glyoxylate) is integrated in the serine cycle (upper part) and is involved in assimilation of acetyl-CoA and regeneration of glyoxylate during growth on C₁ compounds. It is assumed that during growth on C₂ compounds the ethylmalonyl-CoA pathway (Fig. 4) is used exclusively. Dotted lines indicate more than one reaction step.