Reconstitution, morphology and crystallization of a fatty acid β-oxidation multienzyme complex from Pseudomonas fragi

Abstract

The fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the alpha-subunit and the beta-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the alpha- and beta-subunits), the alpha-subunit alone and the beta-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, alpha2beta2, identical with the native HDT oligomeric state from P. fragi, whereas the alpha-subunit and beta-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated alpha- and beta-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the alpha- and beta-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the beta-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the alpha-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the alpha- and beta-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.

Evidence for the physical association of mitochondrial fatty acid oxidation and oxidative phosphorylation complexes

“…Our results indicate that disruption of larger metabolic supercomplexes containing both the ETC and FAO machinery is a viable mechanism for this phenomenon. Mitochondrial FAO has been previously postulated to occur in the context of a multiprotein complex that promotes metabolic channeling, but this has been poorly characterized, and little evidence exists to physically link FAO to the respiratory chain (37)(38)(39)(40).…”

YfcX Enables Medium-Chain-Length Poly(3-Hydroxyalkanoate) Formation from Fatty Acids in Recombinant Escherichia coli fadB Strains

“…These enzymes are often associated with fatty acid ␤-oxidation complexes. Interestingly, YfcY, encoded by a gene located upstream of yfcX, (19), have been shown to copurify in multienzyme complexes composed of multimers of subunits. Since YfcY and YfcX contain activities related to fatty acid degradation, purification of the enzymes coexpressed in strain DH5␣/pYfcYX was performed.…”

Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and Activity Are Regulated by a Sensitive Redox Switch

“…In mammals there are two distinct pathways for ␤-oxidation; very long chain fatty acids and bile acid intermediates are shortened in peroxisomes, whereas medium to long chain mono-and dicarboxylic fatty acids, methyl-branched fatty acids, and prostaglandins are broken down in mitochondria (14). In both mitochondrial and prokaryotic ␤-oxidation systems trifunctional multi-enzyme complexes (comprising MFP and KAT activities) exist allowing for metabolite channeling (15)(16)(17)(18). The crystal structure of the Pseudomonas fragi trifunctional enzyme complex is known and consists of two ␣-subunits (hosting MFP functions; hereafter referred to as PfMFP) and two ␤-subunits (hosting KAT functions; hereafter referred to as PfKAT) in a dynamic complex (16).…”