Malonyl-Coenzyme A Reductase from Chloroflexus aurantiacus, a Key Enzyme of the 3-Hydroxypropionate Cycle for Autotrophic CO2 Fixation

AUTOR(ES)

Hügler, Michael

FONTE

American Society for Microbiology

RESUMO

The 3-hydroxypropionate cycle is a new autotrophic CO2 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 α2 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; Mg2+ and Fe2+ stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent Km 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.

Documentos Relacionados

• Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle
• l-Malyl-Coenzyme A Lyase/β-Methylmalyl-Coenzyme A Lyase from Chloroflexus aurantiacus, a Bifunctional Enzyme Involved in Autotrophic CO2 Fixation
• 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†
• cDNA Cloning, Heterologous Expressions, and Functional Characterization of Malonyl-Coenzyme A:Anthocyanidin 3-O-Glucoside-6"-O-Malonyltransferase from Dahlia Flowers1
• Inhibition of erythromycin synthesis by disruption of malonyl-coenzyme A decarboxylase gene eryM in Saccharopolyspora erythraea.