title: Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation.
abstract: The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.
authors.affiliation: Institut Biologie II, Mikrobiologie, Universität Freiburg, Schänzlestrasse 1, Freiburg D-79104, Germany. alber@uni-freiburg.de
authors.forename: Birgit E
authors.initials: BE
authors.lastname: Alber
authors.forename: Georg
authors.initials: G
authors.lastname: Fuchs
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism*; D019414:Chlorobi / Q000201:enzymology*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000502:physiology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011422:Propionates / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013696:Temperature; D014357:Trypsin / Q000378:metabolism / Q000494:pharmacology
chemical_list: D011422:Propionates; D002245:Carbon Dioxide; D009249:NADP; D000255:Adenosine Triphosphate; D014357:Trypsin; D003066:Coenzyme A Ligases; C068072:propionate - CoA ligase; D008274:Magnesium; D011188:Potassium