Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

Carboxylation activity in M. sedula cell extracts at 55°C. Acetyl-CoA (●) and propionyl-CoA (○) were specifically carboxylated in vitro with ¹⁴CO₂. CO₂ fixation in the presence of both substrates is also shown (▿). The carboxylation reaction with both substrates was inhibited by 1 nM avidin (▾). For assay conditions, see Materials and Methods.

Carboxylation of acetyl-CoA to malonyl-CoA in the presence of ATP and conversion of malonyl-CoA to 3-hydroxypropionate in the presence of NADPH by cell extracts of M. sedula. (A) HPLC analysis of [¹⁴C]malonyl-CoA formed from [¹⁴C]acetyl-CoA and CO₂. The chromatograms were recorded by using a radioactivity monitor calibrated for¹⁴C detection. (I) Control reaction without cell extract after 30 min of incubation. (II) [¹⁴C]malonyl-CoA formation after a 30-min reaction. (III) Carboxylation reaction specifically inhibited with 20 μg of avidin. Retention times: [¹⁴C]acetyl-CoA, 13.3 min; [¹⁴C]malonyl-CoA, 7.5 min. Assay conditions (0.3-ml assay mixture, 1.2-ml headspace): 100 mM Tris-HCl (pH 7.8), 2 mM ATP, 2.5 mM MgCl₂, 3 μmol of KHCO₃, 9.3 nmol of [¹⁴C]acetyl-CoA (18 kBq), 1.5 mg of protein. (B) TLC detection of labelled products formed after carboxylation of [¹⁴C]acetyl-CoA. The solvent system used was butanol-acetic acid-water (12:3:5). The CoA esters present in the samples (4 μl) were hydrolyzed to the free acid form before being loaded onto the TLC plate. Note that acetic acid is volatile under these conditions. Lane 1, carboxylation reaction inhibited with 1 nM avidin. Lane 2, carboxylation of [¹⁴C]acetyl-CoA after a 30-min reaction. Lane 3, formation of 3-hydroxypropionate (3-OHP) after addition of NADPH (0.5 mM) to the reaction in lane 2 and further incubation for 30 min. Assay conditions were as described for panel A. (C) Spectrophotometric assay of the reduction of malonyl-CoA to 3-hydroxypropionate by NADPH. The reaction was started by adding the substrate malonyl-CoA (0.5 mM) as indicated. The dotted lines indicate the initial and final rates of NADPH oxidation.

TLC analysis of labelled products formed by cell extracts of M. sedula from ¹⁴CO₂ and acetyl CoA. (A) Sample (4 μl) not hydrolyzed after 0 (lane 1), 10 (lane 2), and 30 (lane 3) min of incubation. (B) Samples (4 μl) as described for panel A but after alkaline hydrolysis of CoA thioesters. Assay conditions (85-μl assay mixture, 1.4-ml headspace): 100 mM Tris-HCl (pH 7.8), 1 mM ATP, 5 mM MgCl₂, 0.3 mM NADPH, 10 mM KCl, 5 mM DTE, 3 mM acetyl-CoA, 90 nmol of [¹⁴C]Na₂CO₃ (180 kBq), 0.12 mg of protein. The solvent system was as described for Fig. 3. Note that acetic, acrylic, and propionic acids are volatile under these conditions. 3-OHP, 3-hydroxypropionate.

TLC detection of labelled products formed by cell extracts of C. aurantiacus at 45°C from¹⁴CO₂ and acetyl-CoA in the presence of NADPH, after 0 (lane 1), 0.5 (lane 2), 2.5 (lane 3), and 10 (lane 4) min of incubation. The CoA esters in the samples (4 μl) were hydrolyzed by alkali treatment. The solvent system was as described for Fig. 3. Assay conditions (90-μl assay mixture, 1.4-ml headspace): 100 mM Tris-HCl (pH 7.8), 1 mM ATP, 5 mM MgCl₂, 0.3 mM NADPH, 10 mM KCl, 5 mM DTE, 0.1 mM CoA, 90 nmol of [¹⁴C]Na₂CO₃ (180 kBq), 2.7 mM acetyl-CoA, 0.8 mg of protein. 3-OHP, 3-hydroxypropionate. X, unknown product.

Reductive conversion of 3-hydroxypropionate to propionyl-CoA via 3-hydroxypropionyl–CoA in the presence of CoA, ATP, and NADPH by cell extracts of M. sedula. (A) Spectrophotometric recording of NADPH oxidation during the formation of propionyl-CoA from 3-hydroxypropionate. The assay was started by the addition of the substrate as indicated. The dotted line indicates the point at which the reaction rates were determined. For assay conditions, see Materials and Methods. (B) HPLC chromatograms showing the transient formation of the intermediate 3-hydroxypropionyl–CoA (3-OHP-CoA) and the accumulation of the product propionyl-CoA during the reaction in panel A. The samples injected into the column were taken at different reaction times: 0 min, before addition of 3-hydroxypropionate; 5 min, after addition of 3-hydroxypropionate; 12 min, after addition of 3-hydroxypropionate. Control, assay without 3-hydroxypropionate after 12 min of incubation. The chromatograms were recorded with a UV monitor at 260 nm. Retention times: ATP, 3 min; NADP, 4 min; NADPH, 6.5 min; CoASH, 10.5 min; 3-hydroxypropionyl–CoA, 12.5 min; propionyl-CoA, 18.5 min. Note that CoASH and succinyl-CoA have identical retention times. Assay conditions (0.5-ml assay mixture): 100 mM Tris-HCl (pH 7.8), 1 mM 3-hydroxypropionate, 1 mM CoA, 5 mM ATP, 5 mM MgCl₂, 0.5 mM NADPH, 10 mM KCl, 0.5 mg of protein.

Time course of the carboxylation of propionyl-CoA to methylmalonyl-CoA with ¹⁴CO₂ and subsequent conversion to succinate catalyzed by cell extracts of M. sedula at 55°C. (A) HPLC chromatograms showing the incorporation of ¹⁴CO₂ into propionyl-CoA, generating [¹⁴C]methylmalonyl-CoA as transient intermediate. This intermediate is subsequently converted to [¹⁴C]succinate. (B) HPLC chromatograms after alkaline hydrolysis of the CoA esters present in the samples. The labelled compounds in both panels were preliminarily identified by cochromatography with authentic compounds. Assay conditions (1-ml assay mixture, 2-ml headspace): 100 mM Tris-HCl (pH 7.8), 1 mM ATP, 5 mM MgCl₂, 3 mM DTE, 0.5 mM propionyl-CoA, 5 μmol of [¹⁴C]Na₂CO₃ (10 kBq), 0.4 mg of protein.