Publications

• Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity.

  Kiema T.R., Taskinen J.P., Pirilae P.L., Koivuranta K.T., Wierenga R.K., Hiltunen J.K.

  Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of beta-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase- ... >> More

  Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of beta-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase-1/isomerases (H1/I) and (3 S )-hydroxyacyl-CoA dehydrogenases (HAD). Domain A (residues 1-190) comprises the H1/I fold and catalyses both 2-enoyl-CoA hydratase-1 and Delta(3)-Delta(2)-enoyl-CoA isomerase reactions. Domain B (residues 191-280) links domain A to the (3 S )-dehydrogenase region, which includes both domain C (residues 281-474) and domain D (residues 480-583). Domains C and D carry features of the dinucleotide-binding and the dimerization domains of monofunctional HADs respectively. Domain E (residues 584-722) has sequence similarity to domain D of the perMFE-1, which suggests that it has evolved via partial gene duplication. Experiments with engineered perMFE-1 variants demonstrate that the H1/I competence of domain A requires stabilizing interactions with domains D and E. The variant His-perMFE (residues 288-479)Delta, in which the domain C is deleted, is stable and has hydratase-1 activity. It is proposed that the extreme C-terminal domain E in perMFE-1 serves the following three functions: (i) participation in the folding of the N-terminus into a functionally competent H1/I fold, (ii) stabilization of the dehydrogenation domains by interaction with the domain D and (iii) the targeting of the perMFE-1 to peroxisomes via its C-terminal tripeptide. << Less

  Biochem. J. 367:433-441(2002) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Peroxisomal bifunctional protein from rat liver is a trifunctional enzyme possessing 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3, delta 2-enoyl-CoA isomerase activities.

  Palosaari P.M., Hiltunen J.K.

  Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization o ... >> More

  Peroxisomal delta 3, delta 2-enoyl-CoA isomerase (EC 5.3.3.8) was studied in the liver of rats treated with clofibrate. The mitochondrial and peroxisomal isoenzymes were separated chromatographically and the peroxisomal isomerase purified to apparent homogeneity. In addition to the isomerization of 3-enoyl-CoA esters, the purified protein also catalyzed hydration of trans-2-enoyl-CoA and oxidation of L-3-hydroxyacyl-CoA. Incubation of the purified protein with trans-3-decenoyl-CoA, NAD+, and Mg2+ resulted in an increase in absorbance at 303 nm, indicating the formation of 3-ketoacyl-CoA. The protein purified was monomeric, with an estimated molecular weight of 78,000. In immunoblotting it was recognized by the antibody to peroxisomal bifunctional protein from rat liver. Comparison of the amino acid sequences of cyanogen bromide cleaved isomerase with the known sequence of the peroxisomal bifunctional protein from the rat identified them as the same molecule. In control experiments, the peroxisomal bifunctional protein purified according to published methods also catalyzed delta 3, delta 2-enoyl-CoA isomerization. This means that the bifunctional protein of rat liver is in fact a trifunctional enzyme possessing delta 3, delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase (EC 4.2.1.17), and L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities in the same polypeptide. << Less

  J. Biol. Chem. 265:2446-2449(1990) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Delta3,5-delta2,4-dienoyl-CoA isomerase from rat liver. Molecular characterization.

  Filppula S.A., Yagi A.I., Kilpeleainen S.H., Novikov D., Fitzpatrick D.R., Vihinen M., Valle D., Hiltunen J.K.

  rECH1, a recently identified rat cDNA (FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Genomics 27, 457-466) encodes a polypeptide belonging to the hydratase/isomerase superfamily. We modeled the structure of rECH1 based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts th ... >> More

  rECH1, a recently identified rat cDNA (FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Genomics 27, 457-466) encodes a polypeptide belonging to the hydratase/isomerase superfamily. We modeled the structure of rECH1 based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts that rECH1p has the hydratase fold in the core domain and two domains for interaction with other subunits. When we incubated 3,5,8,11, 14-eicosapentaenoyl-CoA with purified rECH1p, the spectral data suggested a switching of the double bonds from the Delta3-Delta5 to the Delta2-Delta4 positions. This was confirmed by demonstrating that the product was a valid substrate for 2,4-dienoyl-CoA reductase. These results indicate that rECH1p is Delta3,5-Delta2,4-dienoyl-CoA isomerase. Subcellular fractionation and immunoelectron microscopy using antibodies to a synthetic polypeptide derived from the C terminus of rECH1p showed that rECH1p is located in the matrix of both mitochondria and peroxisomes in rat liver. Consistent with these observations, the 36,000-Da rECH1p has a potential N-terminal mitochondrial targeting signal as well as a C-terminal peroxisomal targeting signal type 1. Transport of the protein into the mitochondria with cleavage of the targeting signal results in a mature mitochondrial form with a molecular mass of 32,000 Da; transport to peroxisomes yields a protein of 36,000 Da. << Less

  J. Biol. Chem. 273:349-355(1998) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The isomerase and hydratase reaction mechanism of the crotonase active site of the multifunctional enzyme (type-1), as deduced from structures of complexes with 3S-hydroxy-acyl-CoA.

  Kasaragod P., Schmitz W., Hiltunen J.K., Wierenga R.K.

  The multifunctional enzyme, type-1 (MFE1) is involved in several lipid metabolizing pathways. It catalyses: (a) enoyl-CoA isomerase and (b) enoyl-CoA hydratase (EC 4.2.1.17) reactions in its N-terminal crotonase part, as well as (3) a 3S-hydroxy-acyl-CoA dehydrogenase (HAD; EC 1.1.1.35) reaction i ... >> More

  The multifunctional enzyme, type-1 (MFE1) is involved in several lipid metabolizing pathways. It catalyses: (a) enoyl-CoA isomerase and (b) enoyl-CoA hydratase (EC 4.2.1.17) reactions in its N-terminal crotonase part, as well as (3) a 3S-hydroxy-acyl-CoA dehydrogenase (HAD; EC 1.1.1.35) reaction in its C-terminal 3S-hydroxy-acyl-CoA dehydrogenase part. Crystallographic binding studies with rat peroxisomal MFE1, using unbranched and branched 2E-enoyl-CoA substrate molecules, show that the substrate has been hydrated by the enzyme in the crystal and that the product, 3S-hydroxy-acyl-CoA, remains bound in the crotonase active site. The fatty acid tail points into an exit tunnel shaped by loop-2. The thioester oxygen is bound in the classical oxyanion hole of the crotonase fold, stabilizing the enolate reaction intermediate. The structural data of these enzyme product complexes suggest that the catalytic base, Glu123, initiates the isomerase reaction by abstracting the C2-proton from the substrate molecule. Subsequently, in the hydratase reaction, Glu123 completes the catalytic cycle by reprotonating the C2 atom. A catalytic water, bound between the OE1-atoms of the two catalytic glutamates, Glu103 and Glu123, plays an important role in the enoyl-CoA isomerase and the enoyl-CoA hydratase reaction mechanism of MFE1. The structural variability of loop-2 between MFE1 and its monofunctional homologues correlates with differences in the respective substrate preferences and catalytic rates. << Less

  FEBS J. 280:3160-3175(2013) [PubMed] [EuropePMC]