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

Abstract

The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed forC. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus andAcidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula,S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

Carbon dioxide fixation in ‘Archaeoglobus lithotrophicus’: are there multiple autotrophic pathways?

“…Protein was measured according to the Bradford method, using bovine serum albumin as a standard. Biotinylated proteins in cell extracts were detected with peroxidase-conjugated avidin (Menendez et al, 1999) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).…”

Inhibition of acetate and propionate assimilation by itaconate via propionyl-CoA carboxylase in isocitrate lyase-negative purple bacterium Rhodospirillum rubrum

“…The concentrations of propionate and NaHCO 3 were 5 mM. a The rate of endogenous respiration was 25.3 nmol min 31 (mg protein) 31 for P. fulvum and 14.9 nmol min 31 (mg protein) 31 for R. sphaeroides.…”

Stable carbon isotope fractionations of the hyperthermophilic crenarchaeon Metallosphaera sedula

“…2x enriched in 13 C relative to the bulk cell material, which is similar to previous reports for the reductive TCA cycle [15], but di¡erent from C. aurantiacus, a bacterium using the 3-hydroxypropionate pathway, where isoprenoid lipids were depleted in 13 C relative to the bulk cell material. However, M. sedula does not express key enzymes of the reductive TCA cycle [16]. The isotopic di¡erence between isoprenoid lipids of M. sedula and C. aurantiacus may be due to the fact that the isotopic composition of the cell material of C. aurantiacus (ca.…”

“…Metallosphaera sedula, a hyperthermophilic crenarchaeon that can oxidize reduced metal and sulfur species and hydrogen with molecular oxygen as energy source, has been suggested to use a 3-hydroxypropionate-like CO 2 ¢xation pathway [16]. Comparing stable carbon isotopic fractionation in C. aurantiacus with fractionation in M. sedula could provide additional information on the carbon ¢xation pathway used by M. sedula.…”