Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus , a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO 2 Fixation

Abstract

The 3-hydroxypropionate cycle is a new autotrophic CO ₂ fixation pathway in Chloroflexus aurantiacus and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in Chloroflexus aurantiacus . A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H ⁺ → malonate semialdehyde + NADP ⁺ + CoA and malonate semialdehyde + NADPH + H ⁺ → 3-hydroxypropionate + NADP ⁺ . The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an α ₂ composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg ²⁺ and Fe ²⁺ stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent K _m values of 30 μM malonyl-CoA and 25 μM NADPH and a turnover number of 25 s ⁻¹ subunit ⁻¹ . The specific activity in autotrophically grown cells was 0.08 μmol of malonyl-CoA reduced min ⁻¹ (mg of protein) ⁻¹ , compared to 0.03 μmol min ⁻¹ (mg of protein) ⁻¹ in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides.