The biochemistry of peroxisomal beta-oxidation in the yeast Saccharomyces cerevisiae.

Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of beta-oxidation enzyme activities as well as a set of auxiliary activities to provide the beta-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of beta-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p-Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in beta-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in beta-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

The 1.3 A crystal structure of human mitochondrial Delta3-Delta2-enoyl-CoA isomerase shows a novel mode of binding for the fatty acyl group.

The crystal structure of Delta3-Delta2-enoyl-CoA isomerase from human mitochondria (hmEci), complexed with the substrate analogue octanoyl-CoA, has been refined at 1.3 A resolution. This enzyme takes part in the beta-oxidation of unsaturated fatty acids by converting both cis-3 and trans-3-enoyl-CoA esters (with variable length of the acyl group) to trans-2-enoyl-CoA. hmEci belongs to the hydratase/isomerase (crotonase) superfamily. Most of the enzymes belonging to this superfamily are hexamers, but hmEci is shown to be a trimer. The mode of binding of the ligand, octanoyl-CoA, shows that the omega-end of the acyl group binds in a hydrophobic tunnel formed by residues of the loop preceding helix H4 as well as by side-chains of the kinked helix H9. From the structure of the complex it can be seen that Glu136 is the only catalytic residue. The importance of Glu136 for catalysis is confirmed by mutagenesis studies. A cavity analysis shows the presence of two large, adjacent empty hydrophobic cavities near the active site, which are shaped by side-chains of helices H1, H2, H3 and H4. The structure comparison of hmEci with structures of other superfamily members, in particular of rat mitochondrial hydratase (crotonase) and yeast peroxisomal enoyl-CoA isomerase, highlights the variable mode of binding of the fatty acid moiety in this superfamily.

Structural studies on delta(3)-delta(2)-enoyl-CoA isomerase: the variable mode of assembly of the trimeric disks of the crotonase superfamily.

Subunits of the enzymes in the crotonase superfamily form tight trimeric disks. In most members of this protein superfamily these disks assemble further into hexamers. Here we report on the 2.1 A structure of a tight hexameric crystal form of the yeast peroxisomal delta(3)-delta(2)-enoyl-CoA isomerase (Eci1p). A comparison of this structure to a previously solved crystal form of Eci1p and other structures of this superfamily shows that there is much variability with respect to the relative distance between the disks and their relative orientations. In particular helices H2 and H9 are involved in the inter-trimer contacts and there are considerable structural differences in these helices in this superfamily. Helices H2 and H9 are near the catalytic cavity and it is postulated that the observed structural variability of these helices, stabilized by the different modes of assembly, has allowed the evolution of the wide range of substrate and catalytic specificity within this enzyme superfamily.