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 β-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/β-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 β-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 γ-proteobacterium Congregibacter litoralis . This may indicate that part of the reactions may be involved in a different metabolic process.