Malonyl-coenzyme A reductase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO(2) fixation.
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 alpha(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 microM malonyl-CoA and 25 microM NADPH and a turnover number of 25 s(-1) subunit(-1). The specific activity in autotrophically grown cells was 0.08 micromol of malonyl-CoA reduced min(-1) (mg of protein)(-1), compared to 0.03 micromol 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.
Hügler
Michael
M
Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
Menendez
Castor
C
Schägger
Hermann
H
Fuchs
Georg
G
D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000595:Amino Acid Sequence; D002245:Carbon Dioxide / Q000378:metabolism*; D002250:Carbon Radioisotopes; D019414:Chlorobi / Q000201:enzymology*; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D008315:Malondialdehyde / Q000031:analogs & derivatives* / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000032:analysis / Q000378:metabolism*; D010088:Oxidoreductases; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D002245:Carbon Dioxide; D019344:Lactic Acid; D008315:Malondialdehyde; D008316:Malonyl Coenzyme A; D009249:NADP; C039786:malonic semialdehyde; C031601:hydracrylic acid; D010088:Oxidoreductases; C454539:malonyl-Coa reductase; D000426:Alcohol Dehydrogenase; D044943:Fatty Acid Desaturases; D000444:Aldehyde Dehydrogenase