The Experts below are selected from a list of 7971 Experts worldwide ranked by ideXlab platform
J. Kalervo Hiltunen - One of the best experts on this subject based on the ideXlab platform.
-
Crystallographic binding studies of rat peroxisomal Multifunctional Enzyme type 1 with 3-ketodecanoyl-CoA: capturing active and inactive states of its hydratase and dehydrogenase catalytic sites.
Acta Crystallographica Section D Structural Biology, 2020Co-Authors: Shruthi Sridhar, Werner Schmitz, J. Kalervo Hiltunen, Tiila-riikka Kiema, Rajaram Venkatesan, Ulrich Bergmann, R.k. WierengaAbstract:The peroxisomal Multifunctional Enzyme type 1 (MFE1) catalyzes two successive reactions in the β-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD+-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric Enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 A resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD+ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.
-
Peroxisomal Multifunctional Enzyme type 2 from the fruit fly: dehydrogenase and hydratase act as separate entities as revealed by structure and kinetics
Biochemical Journal, 2011Co-Authors: Tatu J. K. Haataja, J. Kalervo Hiltunen, M. Kristian Koski, Tuomo GlumoffAbstract:All the peroxisomal β-oxidation pathways characterized thus far house at least one Multifunctional Enzyme (MFE) catalyzing 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 D. melanogaster MFE-2 (DmMFE-2), complements the S. 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.
-
Peroxisomal Multifunctional Enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics
The Biochemical journal, 2011Co-Authors: Tatu J. K. Haataja, J. Kalervo Hiltunen, M. Kristian Koski, Tuomo GlumoffAbstract: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 ( Dm MFE-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.
-
Crystal Structure of Liganded Rat Peroxisomal Multifunctional Enzyme Type 1 A FLEXIBLE MOLECULE WITH TWO INTERCONNECTED ACTIVE SITES
The Journal of biological chemistry, 2010Co-Authors: Prasad Kasaragod, J. Kalervo Hiltunen, Tiila-riikka Kiema, Rajaram Venkatesan, R.k. WierengaAbstract:Abstract The crystal structure of the full-length rat peroxisomal Multifunctional Enzyme, type 1 (rpMFE1), has been determined at 2.8 A 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 A 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.
-
Crystal Structure of Yeast Peroxisomal Multifunctional Enzyme: Structural Basis for Substrate Specificity of (3R)-hydroxyacyl-CoA Dehydrogenase Units
Journal of molecular biology, 2006Co-Authors: Mari S. Ylianttila, Niko V. Pursiainen, Antti M. Haapalainen, André H. Juffer, Yves Poirier, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:(3R)-hydroxyacyl-CoA dehydrogenase is part of Multifunctional Enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric Multifunctional Enzyme is very different.
Tuomo Glumoff - One of the best experts on this subject based on the ideXlab platform.
-
Peroxisomal Multifunctional Enzyme type 2 from the fruitfly: dehydrogenase and hydratase act as separate entities, as revealed by structure and kinetics
The Biochemical journal, 2011Co-Authors: Tatu J. K. Haataja, J. Kalervo Hiltunen, M. Kristian Koski, Tuomo GlumoffAbstract: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 ( Dm MFE-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.
-
Peroxisomal Multifunctional Enzyme type 2 from the fruit fly: dehydrogenase and hydratase act as separate entities as revealed by structure and kinetics
Biochemical Journal, 2011Co-Authors: Tatu J. K. Haataja, J. Kalervo Hiltunen, M. Kristian Koski, Tuomo GlumoffAbstract:All the peroxisomal β-oxidation pathways characterized thus far house at least one Multifunctional Enzyme (MFE) catalyzing 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 D. melanogaster MFE-2 (DmMFE-2), complements the S. 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.
-
Crystal Structure of Yeast Peroxisomal Multifunctional Enzyme: Structural Basis for Substrate Specificity of (3R)-hydroxyacyl-CoA Dehydrogenase Units
Journal of molecular biology, 2006Co-Authors: Mari S. Ylianttila, Niko V. Pursiainen, Antti M. Haapalainen, André H. Juffer, Yves Poirier, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:(3R)-hydroxyacyl-CoA dehydrogenase is part of Multifunctional Enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric Multifunctional Enzyme is very different.
-
Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal Multifunctional Enzyme type 2.
Journal of molecular biology, 2004Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract: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 b-oxidation of very-long-chain and a-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 3 Aresolution. 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 OC8. 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.
-
Crystallization and preliminary crystallographic data of 2-enoyl-CoA hydratase 2 domain of Candida tropicalis peroxisomal Multifunctional Enzyme type 2.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal Multifunctional Enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.
Antti M. Haapalainen - One of the best experts on this subject based on the ideXlab platform.
-
Crystal Structure of Yeast Peroxisomal Multifunctional Enzyme: Structural Basis for Substrate Specificity of (3R)-hydroxyacyl-CoA Dehydrogenase Units
Journal of molecular biology, 2006Co-Authors: Mari S. Ylianttila, Niko V. Pursiainen, Antti M. Haapalainen, André H. Juffer, Yves Poirier, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:(3R)-hydroxyacyl-CoA dehydrogenase is part of Multifunctional Enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric Multifunctional Enzyme is very different.
-
Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal Multifunctional Enzyme type 2.
Journal of molecular biology, 2004Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract: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 b-oxidation of very-long-chain and a-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 3 Aresolution. 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 OC8. 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.
-
Crystallization and preliminary crystallographic data of 2-enoyl-CoA hydratase 2 domain of Candida tropicalis peroxisomal Multifunctional Enzyme type 2.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:In yeast, the second and the third reaction of the fatty-acid beta-oxidation spiral are catalysed by peroxisomal Multifunctional Enzyme type 2 (Mfe2p/Fox2p). This protein has two (3R)-hydroxyacyl-CoA dehydrogenase domains and a C-terminal 2-enoyl-CoA hydratase 2 domain. Here, the purification, crystallization and X-ray diffraction analysis of the hydratase 2 domain [CtMfe2p(dh(a+b)Delta)] from Candida tropicalis Mfe2p is reported. CtMfe2p(dh(a+b)Delta) was overexpressed as an enzymatically active recombinant protein and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to space group C2, with unit-cell parameters a = 178.57, b = 60.46, c = 130.85 A, beta = 94.48 degrees. Selenomethionine-labelled protein was used for a multi-wavelength anomalous dispersion (MAD) experiment. A three-wavelength data set suitable for MAD phasing was collected to 2.25 A resolution using synchrotron radiation.
-
Binary Structure of the Two-Domain (3R)-Hydroxyacyl-CoA Dehydrogenase from Rat Peroxisomal Multifunctional Enzyme Type 2 at 2.38 Å Resolution
Structure (London England : 1993), 2003Co-Authors: Antti M. Haapalainen, J. Kalervo Hiltunen, Yong-mei Qin, M. Kristian Koski, Tuomo GlumoffAbstract: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.
Journal of molecular biology, 2001Co-Authors: Antti M. Haapalainen, J.k. Hiltunen, R.k. Wierenga, D.m.f. Van Aalten, Gitte Meriläinen, J.e Jalonen, Päivi Pirilä, Tuomo GlumoffAbstract:Abstract β-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 (dΔhΔSCP-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 dΔhΔSCP-2L has an α/β-fold consisting of five β-strands and five α-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of dΔhΔSCP-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.
Yong-mei Qin - One of the best experts on this subject based on the ideXlab platform.
-
Binary Structure of the Two-Domain (3R)-Hydroxyacyl-CoA Dehydrogenase from Rat Peroxisomal Multifunctional Enzyme Type 2 at 2.38 Å Resolution
Structure (London England : 1993), 2003Co-Authors: Antti M. Haapalainen, J. Kalervo Hiltunen, Yong-mei Qin, M. Kristian Koski, Tuomo GlumoffAbstract: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.
-
Human Peroxisomal Multifunctional Enzyme Type 2 SITE-DIRECTED MUTAGENESIS STUDIES SHOW THE IMPORTANCE OF TWO PROTIC RESIDUES FOR 2-ENOYL-CoA HYDRATASE 2 ACTIVITY
The Journal of biological chemistry, 2000Co-Authors: Yong-mei Qin, Antti M. Haapalainen, Tuomo Glumoff, Dmitry K. Novikov, M. Kristian Koski, Mari S. Marttila, Seppo H. Kilpeläinen, J. Kalervo HiltunenAbstract:Abstract β-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 oftrans-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 ak 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.
-
Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis Multifunctional Enzyme.
Journal of lipid research, 2000Co-Authors: Yong-mei Qin, Matti H. Poutanen, Dmitry K. NovikovAbstract: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.
-
Yeast peroxisomal Multifunctional Enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.
The Journal of biological chemistry, 1999Co-Authors: Yong-mei Qin, Antti M. Haapalainen, Tuomo Glumoff, Mari S. Marttila, Kirsi M. Siivari, J. Kalervo HiltunenAbstract:Abstract 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. Gly16 and Gly329 of the S. cerevisiae A and B domains, corresponding to Gly16, 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(aΔ) and pYE352::ScMFE-2(bΔ) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aΔbΔ) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Δ)) and mutational variants of the A and B domains (Ct MFE-2(h2ΔaΔ), Ct MFE- 2(h2ΔbΔ), andCt MFE- 2(h2ΔaΔbΔ)) 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.
-
Peroxisomal Multifunctional Enzyme of beta-oxidation metabolizing D-3-hydroxyacyl-CoA esters in rat liver: molecular cloning, expression and characterization.
Biochemical Journal, 1997Co-Authors: Yong-mei Qin, Matti Poutanen, H M Helander, Ari-pekka Kvist, K M Siivari, W Schmitz, Ernst Conzelmann, Ulf Hellman, J.k. HiltunenAbstract:In the present study we have cloned and characterized a novel rat peroxisomal Multifunctional Enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Makela, Jalonen, Latipaa, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described Multifunctional isomerase-hydratase-dehydrogenase Enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.
Trevor Lithgow - One of the best experts on this subject based on the ideXlab platform.
-
the Multifunctional Enzyme s adenosylhomocysteine methylthioadenosine nucleosidase is a key metabolic Enzyme in the virulence of salmonella enterica var typhimurium
Biochemical Journal, 2019Co-Authors: Asma Ul Husna, Nancy Wang, Jonathan J Wilksch, Hayley J Newton, Dianna M Hocking, Iain D Hay, Simon A Cobbold, Mark R Davies, Malcolm J Mcconville, Trevor LithgowAbstract:Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the Multifunctional Enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.
-
The Multifunctional Enzyme S-adenosylhomocysteine/methylthioadenosine nucleosidase is a key metabolic Enzyme in the virulence of Salmonella enterica var Typhimurium.
The Biochemical journal, 2019Co-Authors: Asma Ul Husna, Nancy Wang, Jonathan J Wilksch, Hayley J Newton, Dianna M Hocking, Iain D Hay, Simon A Cobbold, Mark R Davies, Malcolm J Mcconville, Trevor LithgowAbstract:Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the Multifunctional Enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δpfs). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δpfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δpfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.