Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.     

Structural studies of MFE-1: the 1.9 A crystal structure of the dehydrogenase part of rat peroxisomal MFE-1

The 1.9 A structure of the C-terminal dehydrogenase part of the rat peroxisomal monomeric multifunctional enzyme type 1 (MFE-1) has been determined. In this construct (residues 260-722 and referred to as MFE1-DH) the N-terminal hydratase part of MFE-1 has been deleted. The structure of MFE1-DH shows that it consists of an N-terminal helix, followed by a Rossmann-fold domain (domain C), followed by two tightly associated helical domains (domains D and E), which have similar topology. The structure of MFE1-DH is compared with the two known homologous structures: human mitochondrial 3-hydroxyacyl-CoA dehydrogenase (HAD; sequence identity is 33%) (which is dimeric and monofunctional) and with the dimeric multifunctional alpha-chain (alphaFOM; sequence identity is 28%) of the bacterial fatty acid beta-oxidation alpha2beta2-multienzyme complex. Like MFE-1, alphaFOM has an N-terminal hydratase part and a C-terminal dehydrogenase part, and the structure comparisons show that the N-terminal helix of MFE1-DH corresponds to the alphaFOM linker helix, located between its hydratase and dehydrogenase part. It is also shown that this helix corresponds to the C-terminal helix-10 of the hydratase/isomerase superfamily, suggesting that functionally it belongs to the N-terminal hydratase part of MFE-1.     

Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2

2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I-III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain-length >C8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE-2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed.     

Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme

In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involved in fatty acid and cholesterol side-chain beta-oxidation. Mammalian peroxisomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. As has been shown previously, perMFE-2 hydrates (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24R, 25R)-3alpha, 7alpha,12alpha,24xi-tetrahydroxy-5beta-choles tanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3alpha,7alpha, 12alpha,24xi-tetrahydroxy-5beta-cholestanoyl-CoA , the mammalian perMFE-2 dehydrogenates only the (24R,25R)-isomer. The yeast peroxisomal multifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. To investigate the stereospecificity of yeast peroxisomal MFE, the two dehydrogenase domains of C. tropicalis MFE were expressed in E. coli as a 65 kDa recombinant protein. This protein catalyzes the dehydrogenation of straight-chain (R)-3-hydroxyacyl-CoAs, but it is devoid of (S)-3-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities. The protein dehydrogenates (24R,25R)- and (24R, 25S)-isomers of 3alpha,7alpha, 12alpha, 24xi-tetrahydroxy-5beta-cholestanoyl-CoA. Interestingly, the protein also shows 17beta-estradiol dehydrogenase activity.As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemistry of bile acid synthesis.     

Binary structure of the two-domain (3R)-hydroxyacyl-CoA dehydrogenase from rat peroxisomal multifunctional enzyme type 2 at 2.38 A resolution

The crystal structure of (3R)-hydroxyacyl-CoA dehydrogenase of rat peroxisomal multifunctional enzyme type 2 (MFE-2) was solved at 2.38 A resolution. The catalytic entity reveals an alpha/beta short chain alcohol dehydrogenase/reductase (SDR) fold and the conformation of the bound nicotinamide adenine dinucleotide (NAD(+)) found in other SDR enzymes. Of great interest is the separate COOH-terminal domain, which is not seen in other SDR structures. This domain completes the active site cavity of the neighboring monomer and extends dimeric interactions. Peroxisomal diseases that arise because of point mutations in the dehydrogenase-coding region of the MFE-2 gene can be mapped to changes in amino acids involved in NAD(+) binding and protein dimerization.     

Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A resolution.

beta-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid residues 618-736 of human MFE-2 (d Delta h Delta SCP-2L) was solved at 1.75 A resolution in complex with Triton X-100, an analog of a lipid molecule. This is the first reported structure of an MFE-2 domain. The d Delta h Delta SCP-2L has an alpha/beta-fold consisting of five beta-strands and five alpha-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of d Delta h Delta SCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solvent-exposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.     

Site-directed mutagenesis to enable and improve crystallizability of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase

The N-terminal part of Candida tropicalis MFE-2 (MFE-2(h2Delta)) having two (3R)-hydroxyacyl-CoA dehydrogenases with different substrate specificities has been purified and crystallized as a recombinant protein. The expressed construct was modified so that a stabile, homogeneous protein could be obtained instead of an unstabile wild-type form with a large amount of cleavage products. Cubic crystals with unit cell parameters a=74.895, b=78.340, c=95.445, and alpha=beta=gamma=90 degrees were obtained by using PEG 4000 as a precipitant. The crystals exhibit the space group P2(1)2(1)2(1) and contain one molecule, consisting of two different (3R)-hydroxyacyl-CoA dehydrogenases, in the asymmetric unit. The crystals diffract to a resolution of 2.2A at a conventional X-ray source.     

Spatial arrangement of coenzyme and substrates bound to L-3-hydroxyacyl-CoA dehydrogenase as studied by spin-labeled analogues of NAD+ and CoA.

The synthesis of nitroxide spin-labeled derivatives of S-acetoacetyl-CoA, S-acetoacetylpantetheine, and S-acetoacetylcysteamine is described. These compounds are active substrates of L-3-hydroxyacyl-CoA dehydrogenase [(S)-3-hydroxyacyl-CoA:NAD+ oxidoreductase, EC 1.1.1.35] exhibiting vmax values from 20% to 70% of S-acetoacetyl-CoA itself. S-Acetoacetylpantetheine and S-acetoacetylcysteamine form binary complexes with the enzyme and exhibit ESR spectra typical for immobilized nitroxides. In the case of spin-labeled pantetheine, the radical is more mobile. When spin-labeled substrates are bound simultaneously to each active site of this dimeric enzyme, spin-spin interactions differentiate between two alternate orientations of the substrate [Birktoft, J.J., Holden, H.M., Hamlin, R., Xuong, N.H., & Banaszak, L.J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8262-8266]. The fatty acid moiety is thought to be located in a cleft between two domains whereas a large part of the CoA moiety probably extends into the solution. NAD+, spin-labeled at N6 of the adenine ring, is an active coenzyme of L-3-hydroxyacyl-CoA dehydrogenase (60% vmax). Complexes with the enzyme exhibit ESR spectra typical of highly immobilized nitroxides. Binding of coenzyme NAD+ causes conformational changes of the binary enzyme/substrate complex as revealed by changes in the ESR spectrum of spin-labeled S-acetoacetylpantetheine.     

Multienzyme complexes of fatty acid oxidation from Escherichia coli K12 and from a mutant with a defective L-3-hydroxyacyl coenzyme A dehydrogenase

An Escherichia coli mutant (fadB64), with a defective L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which is unable to grow on long-chain fatty acids as the sole carbon source, was shown to possess a fatty acid oxidation complex that contains five beta-oxidation enzymes, including L-3-hydroxyacyl-CoA dehydrogenase. A comparative study of the complexes from the mutant, from its parental strain and from wild-type E. coli B demonstrated the immunological and gross structural identity of all three fatty acid oxidation complexes. A kinetic evaluation of the complexes led to the suggestion that the mutation may have affected the active site of L-3-hydroxyacyl-CoA dehydrogenase so that it is inactive with acetoacetyl-CoA as a substrate, but exhibits an increasing percentage of the parental dehydrogenase activity with increasing chain length of the substrate.     

A two-domain structure of one subunit explains unique features of eukaryotic hydratase 2

2-Enoyl-CoA hydratase 2, a part from multifunctional enzyme type 2, hydrates trans-2-enoyl-CoA to 3-hydroxyacyl-CoA in the (3R)-hydroxy-dependent route of peroxisomal beta-oxidation of fatty acids. Unliganded and (3R)-hydroxydecanoyl coenzyme A-complexed crystal structures of 2-enoyl-CoA hydratase 2 from Candida tropicalis multifunctional enzyme type 2 were solved to 1.95- and 2.35-A resolution, respectively. 2-Enoyl-CoA hydratase 2 is a dimeric, alpha+beta protein with a novel quaternary structure. The overall structure of the two-domain subunit of eukaryotic 2-enoyl-CoA hydratase 2 resembles the homodimeric, hot dog fold structures of prokaryotic (R)-specific 2-enoyl-CoA hydratase and beta-hydroxydecanoyl thiol ester dehydrase. Importantly, though, the eukaryotic hydratase 2 has a complete hot dog fold only in its C-domain, whereas the N-domain lacks a long central alpha-helix, thus creating space for bulkier substrates in the binding pocket and explaining the observed difference in substrate preference between eukaryotic and prokaryotic enzymes. Although the N- and C-domains have an identity of <10% at the amino acid level, they share a 50% identity at the nucleotide level and fold similarly. We suggest that a subunit of 2-enoyl-CoA hydratase 2 has evolved via a gene duplication with the concomitant loss of one catalytic site. The hydrogen bonding network of the active site of 2-enoyl-CoA hydratase 2 resembles the active site geometry of mitochondrial (S)-specific 2-enoyl-CoA hydratase 1, although in a mirror image fashion. This arrangement allows the reaction to occur by similar mechanism, supported by mutagenesis and mechanistic studies, although via reciprocal stereochemistry.     

Human peroxisomal multifunctional enzyme type 2. Site-directed mutagenesis studies show the importance of two protic residues for 2-enoyl-CoA hydratase 2 activity

Beta-oxidation of acyl-CoAs in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. Amino acid sequence alignment of the 2-enoyl-CoA hydratase 2 domain in human MFE-2 with other MFE-2s reveals conserved protic residues: Tyr-347, Glu-366, Asp-370, His-406, Glu-408, Tyr-410, Asp-490, Tyr-505, Asp-510, His-515, Asp-517, and His-532. To investigate their potential roles in catalysis, each residue was replaced by alanine in site-directed mutagenesis, and the resulting constructs were tested for complementation in a yeast. After additional screening, the wild type and noncomplementing E366A and D510A variants were expressed and characterized. The purified proteins have similar secondary structural elements, with the same subunit composition. The E366A variant had a k(cat)/K(m) value 100 times lower than that of the wild type MFE-2 at pH 5, whereas the D510A variant was inactive. Asp-510 was imbedded in a novel hydratase 2 motif found in the hydratase 2 proteins. The data show that the hydratase 2 reaction catalyzed by MFE-2 requires two protic residues, Glu-366 and Asp-510, suggesting that their catalytic role may be equivalent to that of the two catalytic residues of hydratase 1.     

Peroxisomal multifunctional enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics

All of the peroxisomal β-oxidation pathways characterized thus far house at least one MFE (multifunctional enzyme) catalysing two out of four reactions of the spiral. MFE type?2 proteins from various species display great variation in domain composition and predicted substrate preference. The gene CG3415 encodes for Drosophila melanogaster MFE-2 (DmMFE-2), complements the Saccharomyces cerevisiae MFE-2 deletion strain, and the recombinant protein displays both MFE-2 enzymatic activities in vitro. The resolved crystal structure is the first one for a full-length MFE-2 revealing the assembly of domains, and the data can also be transferred to structure-function studies for other MFE-2 proteins. The structure explains the necessity of dimerization. The lack of substrate channelling is proposed based on both the structural features, as well as by the fact that hydration and dehydrogenation activities of MFE-2, if produced as separate enzymes, are equally efficient in catalysis as the full-length MFE-2.     

Human liver long-chain 3-hydroxyacyl-coenzyme A dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria.

We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71 kDa and 47 kDa within a native structure of 230 kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C10 to C16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C4 substrates but all are active with C6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.     

Knocking out the MFE-2 gene of Candida bombicola leads to improved medium-chain sophorolipid production

The nonpathogenic yeast Candida bombicola synthesizes sophorolipids. These biosurfactants are composed of the disaccharide sophorose linked to a long-chain hydroxy fatty acid and have potential applications in the food, pharmaceutical, cosmetic and cleaning industries. In order to expand the range of application, a shift of the fatty acid moiety towards medium-chain lengths would be recommendable. However, the synthesis of medium-chain sophorolipids by C. bombicola is a challenging objective. First of all, these sophorolipids can only be obtained by fermentations on unconventional carbon sources, which often have a toxic effect on the cells. Furthermore, medium-chain substrates are partially metabolized in the β-oxidation pathway. In order to redirect unconventional substrates towards sophorolipid synthesis, the β-oxidation pathway was blocked on the genome level by knocking out the multifunctional enzyme type 2 (MFE-2) gene. The total gene sequence of the C. bombicola MFE-2 (6033 bp) was cloned (GenBank accession number EU371724 ), and the obtained nucleotide sequence was used to construct a knock-out cassette. Several knock-out mutants with the correct geno- and phenotype were evaluated in a fermentation on 1-dodecanol. All mutants showed a 1.7–2.9 times higher production of sophorolipids, indicating that in those strains the substrate is redirected towards the sophorolipid synthesis.     

Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the fox2 gene and gene product.

The gene encoding the multifunctional protein (MFP) of peroxisomal beta-oxidation in Saccharomyces cerevisiae was isolated from a genomic library via functional complementation of a fox2 mutant strain. The open reading frame consists of 2700 base pairs encoding a protein of 900 amino acids. The predicted molecular weight (98,759) is in close agreement with that of the isolated polypeptide (96,000). Analysis of the deduced amino acid sequence revealed similarity to the MFPs of two other fungi but not to that of rat peroxisomes or the multifunctional subunit of the Escherichia coli beta-oxidation complex. The FOX2 gene was overexpressed from a multicopy vector (YEp352) in S. cerevisiae and the gene product purified to apparent homogeneity. A truncated version of MFP lacking 271 carboxyl-terminal amino acids was also overexpressed and purified. Experiments to study the enzymatic properties of the wild-type MFP demonstrated an absence of activities originally assigned to an MFP of S. cerevisiae (crotonase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase), whereas two other activities were found: 2-enoyl-CoA hydratase 2 (converting trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA) and D-3-hydroxyacyl CoA dehydrogenase (converting D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA). The truncated form contained only the D-3-hydroxyacyl-CoA dehydrogenase activity. These results clearly demonstrate that the beta-oxidation of fatty acids in S. cerevisiae follows a previously unknown stereochemical course, namely it occurs via a D-3-hydroxyacyl-CoA intermediate.     

Crystal Structure of Liganded Rat Peroxisomal Multifunctional Enzyme Type 1: A FLEXIBLE MOLECULE WITH TWO INTERCONNECTED ACTIVE SITES*

The crystal structure of the full-length rat peroxisomal multifunctional enzyme, type 1 (rpMFE1), has been determined at 2.8 Å resolution. This enzyme has three catalytic activities and two active sites. The N-terminal part has the crotonase fold, which builds the active site for the Δ³,Δ²-enoyl-CoA isomerase and the Δ²-enoyl-CoA hydratase-1 catalytic activities, and the C-terminal part has the (3S)-hydroxyacyl-CoA dehydrogenase fold and makes the (3S)-hydroxyacyl-CoA dehydrogenase active site. rpMFE1 is a multidomain protein having five domains (A–E). The crystal structure of full-length rpMFE1 shows a flexible arrangement of the A-domain with respect to the B–E-domains. Because of a hinge region near the end of the A-domain, two different positions of the A-domain were observed for the two protein molecules (A and B) of the asymmetric unit. In the most closed conformation, the mode of binding of CoA is stabilized by domains A and B (helix-10), as seen in other crotonase fold members. Domain B, although functionally belonging to the N-terminal part, is found tightly associated with the C-terminal part, i.e. fixed to the E-domain. The two active sites of rpMFE1 are ∼40 Å apart, separated by a tunnel, characterized by an excess of positively charged side chains. Comparison of the structures of rpMFE1 with the monofunctional crotonase and (3S)-hydroxyacyl-CoA dehydrogenase superfamily enzymes, as well as with the bacterial α₂β₂-fatty acid oxidation multienzyme complex, reveals that this tunnel could be important for substrate channeling, as observed earlier on the basis of the kinetics of rpMFE1 purified from rat liver.     

Effect of dietary alpha-linolenic acid on the activity and gene expression of hepatic fatty acid oxidation enzymes

The activities of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) were compared with those in the animals fed safflower oil rich in linoleic acid (18:2) and saturated fats (coconut or palm oil). Mitochondrial and peroxisomal palmitoyl-CoA (16:0-CoA) oxidation rates in the liver homogenates were significantly higher in rats fed linseed and perilla oils than in those fed saturated fats and safflower oil. The fatty oxidation rates increased as dietary levels of alpha-18:3 increased. Dietary alpha-18:3 also increased the activity of fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. Unexpectedly, dietary alpha-18:3 caused great reduction in the activity of 3-hydroxyacyl-CoA dehydrogenase measured with short- and medium-chain substrates but not with long-chain substrate. Dietary alpha-18:3 significantly increased the mRNA levels of hepatic fatty acid oxidation enzymes including carnitine palmitoyltransferase I and II, mitochondrial trifunctional protein, acyl-CoA oxidase, peroxisomal bifunctional protein, mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, 2, 4-dienoyl-CoA reductase and delta3, delta2-enoyl-CoA isomerase. Fish oil rich in very long-chain n-3 fatty acids caused similar changes in hepatic fatty acid oxidation. Regarding the substrate specificity of beta-oxidation pathway, mitochondrial and peroxisomal beta-oxidation rate of alpha-18:3-CoA, relative to 16:0- and 18:2-CoAs, was higher irrespective of the substrate/albumin ratios in the assay mixture or dietary fat sources. The substrate specificity of carnitine palmitoyltransferase I appeared to be responsible for the differential mitochondrial oxidation rates of these acyl-CoA substrates. Dietary fats rich in alpha-18:3-CoA relative to safflower oil did not affect the hepatic activity of fatty acid synthase and glucose 6-phosphate dehydrogenase. It was suggested that both substrate specificities and alterations in the activities of the enzymes in beta-oxidation pathway play a significant role in the regulation of the serum lipid concentrations in rats fed alpha-18:3.     

On the Molecular Basis of D-Bifunctional Protein Deficiency Type III

Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K_m, V_max and k_cat) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.     

The Multifunctional Protein in Peroxisomal β-Oxidation: STRUCTURE AND SUBSTRATE SPECIFICITY OF THE ARABIDOPSIS THALIANA PROTEIN MFP2*

Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal β-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.