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Tuomo Glumoff - One of the best experts on this subject based on the ideXlab platform.
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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.
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A Two-domain Structure of One Subunit Explains Unique Features of Eukaryotic Hydratase 2.
The Journal of biological chemistry, 2004Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:Abstract 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 β-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, α+β 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 β-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 α-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
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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.
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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.
Peter J Tonge - One of the best experts on this subject based on the ideXlab platform.
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Effect of mutagenesis on the stereochemistry of Enoyl-CoA Hydratase.
Biochemistry, 2002Co-Authors: Yuguo Feng, Hilary A. Hofstein, Jacque Zwahlen, Peter J TongeAbstract:Enoyl-CoA Hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)-HB-CoA, 3(S)-hydroxybutyryl-CoA with a stereospecificity (kS/kR) of 400000 to 1 [Wu, W. J., Feng, Y., He, X., Hofstein, H. S., Raleigh, D. P., and Tonge, P. J. (2000) J. Am. Chem. Soc. 122, 3987−3994]. Replacement of E164, one of the catalytic glutamates in the active site, with either aspartate or glutamine reduces the rate of formation of the 3(S) product enantiomer (kS) without affecting the rate of formation of the 3(R) product (kR). Consequently, kS/kR is 1000 and 0.33 for E164D and E164Q, respectively. In contrast, mutagenesis of E144, the second catalytic glutamate, reduces the rate of formation of both product enantiomers. Thus, only E144 is required for the formation of 3(R)-HB-CoA, 3(R)-hydroxybutyryl-CoA. Modeling studies together with analysis of α-proton exchange rates and experiments with crotonyl-oxyCoA, a substrate analogue in which the α-proton acidity has been reduced 10000-fold, support a mechanism of 3(R)-hydro...
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Stereoselectivity of Enoyl-CoA Hydratase results from preferential activation of one of two bound substrate conformers.
Chemistry & biology, 2002Co-Authors: Alasdair F. Bell, Yuguo Feng, Hilary A. Hofstein, Sapan Parikh, Michael J. Rudolph, Caroline Kisker, Adrian Whitty, Peter J TongeAbstract:Enoyl-CoA Hydratase catalyzes the hydration of trans-2-crotonyl-CoA to 3(S)- and 3(R)-hydroxybutyryl-CoA with a stereoselectivity (3(S)/3(R)) of 400,000 to 1. Importantly, Raman spectroscopy reveals that both the s-cis and s-trans conformers of the substrate analog hexadiEnoyl-CoA are bound to the enzyme, but that only the s-cis conformer is polarized. This selective polarization is an example of ground state strain, indicating the existence of catalytically relevant ground state destabilization arising from the selective complementarity of the enzyme toward the transition state rather than the ground state. Consequently, the stereoselectivity of the enzyme-catalyzed reaction results from the selective activation of one of two bound substrate conformers rather than from selective binding of a single conformer. These findings have important implications for inhibitor design and the role of ground state interactions in enzyme catalysis.
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Involvement of glycine 141 in substrate activation by Enoyl-CoA Hydratase.
Biochemistry, 2001Co-Authors: Alasdair F. Bell, Yuguo Feng, Peter J TongeAbstract:Raman spectroscopy has been used to investigate the structure of a substrate analogue, hexadiEnoyl-CoA (HD-CoA), bound to wild-type Enoyl-CoA Hydratase and G141P, a mutant in which a hydrogen bond ...
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Stereospecificity of the Reaction Catalyzed by Enoyl-CoA Hydratase
Journal of the American Chemical Society, 2000Co-Authors: Yuguo Feng, Hilary A. Hofstein, Daniel P. Raleigh, Peter J TongeAbstract:Enoyl-CoA Hydratase catalyzes the stereospecific hydration of α,β-unsaturated acyl-CoA thiolesters. Hydration of trans-2-crotonyl-CoA to 3(S)-hydroxybutyryl-CoA proceeds via the syn addition of water and thus the pro-2R proton of 3(S)-hydroxybutyryl-CoA is derived from solvent. Incubation of 3(S)-hydroxybutyryl-CoA with enzyme in D2O results in the slow exchange of the pro-2S proton with solvent deuterium, in addition to the anticipated rapid exchange of the pro-2R proton. Further experiments have shown that the exchange of the pro-2S proton occurs in concert with the formation of the incorrect 3(R)-hydroxybutyryl-CoA enantiomer. The rate of 3(R)-hydroxybutyryl-CoA formation is 4 × 105-fold slower than the normal hydration reaction, but at least 1.6 × 106-fold faster than the non-enzyme-catalyzed reaction. This has allowed us to determine that the absolute stereospecificity for the enzyme-catalyzed reaction is 1 in 4 × 105. The initial formation of 3(R)-hydroxybutyryl-CoA is hypothesized to occur via the ...
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Role of glutamate 144 and glutamate 164 in the catalytic mechanism of Enoyl-CoA Hydratase.
Biochemistry, 1999Co-Authors: Hilary A. Hofstein, Yuguo Feng, Vernon E Anderson, Peter J TongeAbstract:The role of two glutamate residues (E164 and E144) in the active site of Enoyl-CoA Hydratase has been probed by site-directed mutagenesis. The catalytic activity of the E164Q and E144Q mutants has been determined using 3‘-dephosphocrotonyl-CoA. Removal of the 3‘-phosphate group reduces the affinity of the substrate for the enzyme, thereby facilitating the determination of Km and simplifying the analysis of the enzymes' pH dependence. kcat for the hydration of 3‘-dephosphocrotonyl-CoA is reduced 7700-fold for the E144Q mutant and 630000-fold for the E164Q mutant, while Km is unaffected. These results indicate that both glutamate residues play crucial roles in the hydration chemistry catalyzed by the enzyme. Previously, we reported that, in contrast to the wild-type enzyme, the E164Q mutant was unable to exchange the α-proton of butyryl-CoA with D2O [D‘Ordine, R. L., Bahnson, B. J., Tonge, P. J., and Anderson, V. E. (1994) Biochemistry 33, 14733−14742]. Here we demonstrate that E144Q is also unable to catal...
J. Kalervo Hiltunen - One of the best experts on this subject based on the ideXlab platform.
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Identification and functional characterization of a monofunctional peroxisomal Enoyl-CoA Hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.
The Journal of biological chemistry, 2006Co-Authors: Simon Goepfert, J. Kalervo Hiltunen, Yves PoirierAbstract:A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal Enoyl-CoA Hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-Enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have Enoyl-CoA Hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-Enoyl-CoA for further degradation through the core beta-oxidation cycle.
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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.
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A Two-domain Structure of One Subunit Explains Unique Features of Eukaryotic Hydratase 2.
The Journal of biological chemistry, 2004Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:Abstract 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 β-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, α+β 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 β-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 α-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
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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.
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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.
Rik K. Wierenga - One of the best experts on this subject based on the ideXlab platform.
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Mutagenic and enzymological studies of the Hydratase and isomerase activities of 2-Enoyl-CoA Hydratase-1.
Biochemistry, 1999Co-Authors: Tiila-riikka Kiema, Rik K. Wierenga, C. K. Engel, Schmitz W, S.a. Filppula, J. K. HiltunenAbstract:Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-Enoyl-CoA Hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-Enoyl-CoA Hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Δ3-Δ2-Enoyl-CoA isomerase activity of mECH-1, a new assay using 2-Enoyl-CoA Hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively....
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Crystallization experiments with 2-Enoyl-CoA Hydratase, using an automated 'fast-screening' crystallization protocol.
Acta Crystallographica Section D Biological Crystallography, 1994Co-Authors: J. P. Zeelen, J. K. Hiltunen, T. A. Ceska, Rik K. WierengaAbstract:A convenient method for screening crystallization conditions using an automated fast-screen protocol has been implemented and tested on an Enoyl-CoA Hydratase. The crystallization solutions for the initial screening and subsequent optimizations are prepared using a crystallization robot. Enoyl-CoA Hydratase (E.C. 4.2.1.17), purified from rat-liver mitochondria, is one of the enzymes from the β-oxidation pathway of fatty-acid metabolism; it catalyzes the reversible hydration of 2-trans-Enoyl-CoA's to l-3-hydroxy-acyl-CoA's. Different crystal forms, diffracting to 3.0 A, were obtained.
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Crystallization experiments with 2-Enoyl-CoA Hydratase, using an automated 'fast-screening' crystallization protocol.
Acta crystallographica. Section D Biological crystallography, 1994Co-Authors: J. P. Zeelen, J. K. Hiltunen, T. A. Ceska, Rik K. WierengaAbstract:A convenient method for screening crystallization conditions using an automated fast-screen protocol has been implemented and tested on an Enoyl-CoA Hydratase. The crystallization solutions for the initial screening and subsequent optimizations are prepared using a crystallization robot. Enoyl-CoA Hydratase (E.C. 4.2.1.17), purified from rat-liver mitochondria, is one of the enzymes from the beta-oxidation pathway of fatty-acid metabolism; it catalyzes the reversible hydration of 2-trans-Enoyl-CoA's to L-3-hydroxy-acyl-CoA's. Different crystal forms, diffracting to 3.0 A, were obtained.
M. Kristian Koski - One of the best experts on this subject based on the ideXlab platform.
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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.
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A Two-domain Structure of One Subunit Explains Unique Features of Eukaryotic Hydratase 2.
The Journal of biological chemistry, 2004Co-Authors: M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen, Tuomo GlumoffAbstract:Abstract 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 β-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, α+β 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 β-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 α-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
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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.
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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.