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Journal of Bacteriology, May 2002, p. 2404-2410, Vol. 184, No. 9
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.9.2404-2410.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Michael Hügler,1 Castor Menendez,1 Hermann Schägger,2 and Georg Fuchs1*

Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg, Freiburg,1 Zentrum der Biologischen Chemie, Klinikum der Johann-Wolfgang-Goethe-Universität Frankfurt, Frankfurt, Germany2

Received 29 October 2001/ Accepted 7 February 2002

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 {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; 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.

* Corresponding author. Mailing address: Mikrobiologie, Institut Biologie II, Schänzlestrasse 1, D-79104 Freiburg, Germany. Phone: 49-761-2032649. Fax: 49-761-2032626. E-mail: fuchsgeo{at}uni-freiburg.de.

Journal of Bacteriology, May 2002, p. 2404-2410, Vol. 184, No. 9
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.9.2404-2410.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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