Structural basis for channelling mechanism of a fatty acid β-oxidation multienzyme complex

Abstract

The atomic view of the active site coupling termed channelling is a major subject in molecular biology. We have determined two distinct crystal structures of the bacterial multienzyme complex that catalyzes the last three sequential reactions in the fatty acid b-oxidation cycle. The a 2 b 2 heterotetrameric structure shows the uneven ring architecture, where all the catalytic centers of 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) face a large inner solvent region. The substrate, anchored through the 3 0 -phosphate ADP moiety, allows the fatty acid tail to pivot from the ECH to HACD active sites, and finally to the KACT active site. Coupling with striking domain rearrangements, the incorporation of the tail into the KACT cavity and the relocation of 3 0 -phosphate ADP bring the reactive C2-C3 bond to the correct position for cleavage. The ahelical linker specific for the multienzyme contributes to the pivoting center formation and the substrate transfer through its deformation. This channelling mechanism could be applied to other b-oxidation multienzymes, as revealed from the homology model of the human mitochondrial trifunctional enzyme complex.

The Multifunctional Protein in Peroxisomal β-Oxidation

“…Amino acid residues observed to hydrogen bond to the cofactor in monofunctional HACDs (67,68) and PfMFP are conserved and have similar conformations in AtMFP2-HACD (Glu 342 , Glu 401 , Lys 406 , and Asn 624 ), but no significant electron density is observed in the AtMFP2 co-factor-binding site. A similar absence of positive electron density for the co-factor is observed in the crystal structure of the truncated recombinant RnMFE-1-HACD (15), whereas NAD ϩ is present in the active site of the PfMFP ␤-oxidation complex but with an average B-factor of 98 Å 2 (13). As evidenced by the enzyme activity characterization included in this study, the AtMFP2 used for crystallization experiments is an active enzyme transferring electrons to NAD ϩ .…”

“…In the yeast lipolytica, the five ACX isoforms present form a heteropentameric complex and are imported into the peroxisomes as such (12). Bacterial and mammalian mitochondrial multifunctional complexes include ECH, HACD, and KAT activities (13,14). Monofunctional enoyl-CoA isomerases/hydratases, HACD, and KAT, with a preference for shorter chain length substrates, are present in mammalian mitochondria in addition to the MFPs.…”

“…The part of the acyl-binding pocket that is included in the structure of AtMFP2-ECH is dominated by hydrophobic residues (Phe 33 ). An MFP channeling mechanism, with the nucleotide being the pivot shaft for transfer of the acyl chain from ECH to the HACD active site, has been suggested in bacteria (13). If channeling is indeed a common phenomenon among the MFPs, the proposed nucleotide environment of the MFPs is expected to be conserved and would likely include an extended conformation of the acyl-CoA in order to bridge the 40 Å span from the ECH to the HACD active site.…”

Crystal Structure of the ECH2 Catalytic Domain of CurF from Lyngbya majuscula

“…S1). Thus, the ECH 2 domain may be monomeric in the context of full-length CurF, similar to the bacterial fatty acid ␤-oxidation multienzyme complex in which an N-terminal monomeric crotonase domain is fused to a dimeric dehydrogenase domain (50). Nevertheless, existence of the classical crotonase trimer in the isolated ECH 2 domain (Fig.…”

Production of renewable polymers from crop plants

“…The differences in the b-oxidation pathway that accounts for this discrepancy are not known, but could include the presence of enzymes such as a 3-hydroxyacyl-CoA epimerase, enabling more efficient synthesis of the PHA substrate R-3-hydroxyacyl-CoAs from the b-oxidation intermediate S-3-hydroxyacyl-CoAs. Furthermore, metabolic channeling of intermediates of b-oxidation has been observed for the rat mitochondrial b-oxidation pathway, and the presence of similar channeling in peroxisomal b-oxidation may well strongly limit the availability of the intermediate 3-hydroxyacyl-CoA to the PHA synthase (Ishikawa et al, 2004). From the experience gathered in targeting the PHA pathway to various subcellular organelles, the best location in terms of PHA quantity appears to be the plastid, although clearly more work needs to be done to understand how synthesis of the precursors (e.g.…”