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Rik K. Wierenga - One of the best experts on this subject based on the ideXlab platform.
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the peroxisomal zebrafish scp2 Thiolase type 1 is a weak transient dimer as revealed by crystal structures and native mass spectrometry
Biochemical Journal, 2019Co-Authors: Tiila-riikka Kiema, Toshiyuki Fukao, Werner Schmitz, C J Thapa, Mikko Laitaoja, Mirko M Maksimainen, Juha Rouvinen, Janne Janis, Rik K. WierengaAbstract:The SCP2 (sterol carrier protein 2)-Thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the β-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric Thiolases has previously been poorly characterized. High-resolution crystal structures of the zebrafish SCP2-Thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerized monomeric form has also been captured in the obtained crystals. Four loops at the dimer interface adopt very different conformations in the monomeric form. These loops also shape the active site and their structural changes explain why a competent active site is not present in the monomeric form. Native mass spectrometry studies confirm that the zebrafish SCP2-Thiolase (type-1) as well as its human homolog are weak transient dimers in solution. The crystallographic binding studies reveal the mode of binding of CoA and octanoyl-CoA in the active site, highlighting the conserved geometry of the nucleophilic cysteine, the catalytic acid/base cysteine and the two oxyanion holes. The dimer interface of SCP2-Thiolase (type-1) is equally extensive as in other Thiolase dimers; however, it is more polar than any of the corresponding interfaces, which correlates with the notion that the enzyme forms a weak transient dimer. The structure comparison of the monomeric and dimeric forms suggests functional relevance of this property. These comparisons provide also insights into the structural rearrangements that occur when the folded inactive monomers assemble into the mature dimer.
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Crystallographic substrate binding studies of Leishmania mexicana SCP2-Thiolase (type-2): unique features of oxyanion hole-1.
Protein Engineering Design & Selection, 2017Co-Authors: R K Harijan, Paul A M Michels, Tiila-riikka Kiema, Shahan M. Syed, Imran Qadir, Muriel Mazet, Frédéric Bringaud, Rik K. WierengaAbstract:Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-Thiolase (type-2) (Lm-Thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of Thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-Thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-Thiolases, whereas SCP2-Thiolases (type-1, type-2) are classified as CHH-Thiolases, having as corresponding fingerprints xS, DCF and G P. In all Thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-Thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-Thiolase structures, it is seen that in this CHH-Thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.
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Crystal structure of a Thiolase from Escherichia coli at 1.8 angstrom resolution
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2016Co-Authors: M. Ithayaraja, Rik K. Wierenga, Handanahal S. Savithri, Neelanjana Janardan, M. R N MurthyAbstract:Thiolases catalyze the Claisen condensation of two acetyl-CoA molecules to give acetoacetyl-CoA, as well as the reverse degradative reaction. Four genes coding for Thiolases or Thiolase-like proteins are found in the Escherichia coli genome. In this communication, the successful cloning, purification, crystallization and structure determination at 1.8 angstrom resolution of a homotetrameric E. coli Thiolase are reported. The structure of E. coli Thiolase co-crystallized with acetyl-CoA at 1.9 angstrom resolution is also reported. As observed in other tetrameric Thiolases, the present E. coli Thiolase is a dimer of two tight dimers and probably functions as a biodegradative enzyme. Comparison of the structure and biochemical properties of the E. coli enzyme with those of other well studied Thiolases reveals certain novel features of this enzyme, such as the modification of a lysine in the dimeric interface, the possible oxidation of the catalytic Cys88 in the structure of the enzyme obtained in the presence of CoA and active-site hydration. The tetrameric enzyme also displays an interesting departure from exact 222 symmetry, which is probably related to the deformation of the tetramerization domain that stabilizes the oligomeric structure of the protein. The current study allows the identification of substrate-binding amino-acid residues and water networks at the active site and provides the structural framework required for understanding the biochemical properties as well as the physiological function of this E. coli Thiolase.
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Crystal structure of a Thiolase from Escherichia coli at 1.8Å resolution
Acta Crystallographica Section:F Structural Biology Communications, 2016Co-Authors: M. Ithayaraja, N. Janardan, Rik K. Wierenga, Handanahal S. Savithri, M. R N MurthyAbstract:Thiolases catalyze the Claisen condensation of two acetyl-CoA molecules to give acetoacetyl-CoA, as well as the reverse degradative reaction. Four genes coding for Thiolases or Thiolase-like proteins are found in the Escherichia coli genome. In this communication, the successful cloning, purification, crystallization and structure determination at 1.8 Å resolution of a homotetrameric E. coli Thiolase are reported. The structure of E. coli Thiolase co-crystallized with acetyl-CoA at 1.9 Å resolution is also reported. As observed in other tetrameric Thiolases, the present E. coli Thiolase is a dimer of two tight dimers and probably functions as a biodegradative enzyme. Comparison of the structure and biochemical properties of the E. coli enzyme with those of other well studied Thiolases reveals certain novel features of this enzyme, such as the modification of a lysine in the dimeric interface, the possible oxidation of the catalytic Cys88 in the structure of the enzyme obtained in the presence of CoA and active-site hydration. The tetrameric enzyme also displays an interesting departure from exact 222 symmetry, which is probably related to the deformation of the tetramerization domain that stabilizes the oligomeric structure of the protein. The current study allows the identification of substrate-binding amino-acid residues and water networks at the active site and provides the structural framework required for understanding the biochemical properties as well as the physiological function of this E. coli Thiolase.
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the crystal structure of human mitochondrial 3 ketoacyl coa Thiolase t1 insight into the reaction mechanism of its Thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, R K Harijan, Toshiyuki Fukao, Malgorzata Strozyk, Stefan E H Alexson, Rik K. WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA Thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative Thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric Thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative Thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic Thiolase activity, which is predicted to be of physiological importance.
Richard A Rachubinski - One of the best experts on this subject based on the ideXlab platform.
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a role for the peroxin pex8p in pex20p dependent Thiolase import into peroxisomes of the yeast yarrowia lipolytica
Journal of Biological Chemistry, 2001Co-Authors: Jennifer J Smith, Richard A RachubinskiAbstract:Abstract Peroxins are proteins required for peroxisome assembly. The cytosolic peroxin Pex20p binds directly to the β-oxidation enzyme Thiolase and is necessary for its dimerization and peroxisomal targeting. The intraperoxisomal peroxin Pex8p has a role in the import of peroxisomal matrix proteins, including Thiolase. We report the results of yeast two-hybrid analyses with various peroxins of the yeast Yarrowia lipolytica and characterize more fully the interaction between Pex8p and Pex20p. Coimmunoprecipitation showed that Pex8p and Pex20p form a complex, while in vitrobinding studies demonstrated that the interaction between Pex8p and Pex20p is specific, direct, and autonomous. Pex8p fractionates with peroxisomes in cells of a PEX20 disruption strain, indicating that Pex20p is not necessary for the targeting of Pex8p to peroxisomes. In cells of a PEX8 disruption strain, Thiolase is mostly cytosolic, while Pex20p and a small amount of Thiolase associate with peroxisomes, suggesting the involvement of Pex8p in the import of Thiolase after docking of the Pex20p-Thiolase complex to the membrane. In the absence of Pex8p, peroxisomal Thiolase and Pex20p are protected from the action of externally added protease. This finding, together with the fact that Pex8p is intraperoxisomal, suggests that Pex20p may accompany Thiolase into peroxisomes during import.
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pex20p of the yeast yarrowia lipolytica is required for the oligomerization of Thiolase in the cytosol and for its targeting to the peroxisome
Journal of Cell Biology, 1998Co-Authors: Vladimir I Titorenko, Jennifer J Smith, Rachel K Szilard, Richard A RachubinskiAbstract:Pex mutants are defective in peroxisome assembly. In the pex20-1 mutant strain of the yeast Yarrowia lipolytica, the peroxisomal matrix protein Thiolase is mislocalized exclusively to the cytosol, whereas the import of other peroxisomal proteins is unaffected. The PEX20 gene was isolated by functional complementation of the pex20-1 strain and encodes a protein, Pex20p, of 424 amino acids (47,274 D). Despite its role in the peroxisomal import of Thiolase, which is targeted by an amino-terminal peroxisomal targeting signal-2 (PTS2), Pex20p does not exhibit homology to Pex7p, which acts as the PTS2 receptor. Pex20p is mostly cytosolic, whereas 4–8% is associated with high-speed (200,000 g) pelletable peroxisomes. In the wild-type strain, all newly synthesized Thiolase is associated with Pex20p in a heterotetrameric complex composed of two polypeptide chains of each protein. This association is independent of PTS2. Pex20p is required for both the oligomerization of Thiolase in the cytosol and its targeting to the peroxisome. Our data suggest that monomeric Pex20p binds newly synthesized monomeric Thiolase in the cytosol and promotes the formation of a heterotetrameric complex of these two proteins, which could further bind to the peroxisomal membrane. Translocation of the Thiolase homodimer into the peroxisomal matrix would release Pex20p monomers back to the cytosol, thereby permitting a new cycle of binding-oligomerization-targeting-release for Pex20p and Thiolase.
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saccharomyces cerevisiae peroxisomal Thiolase is imported as a dimer
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: J R Glover, Davidw . Andrews, Richard A RachubinskiAbstract:Abstract The active conformation of native peroxisomal 3-ketoacyl-CoA Thiolases (EC 2.3.1.16) is homodimeric. We have previously shown that a truncated Saccharomyces cerevisiae Thiolase lacking its first 16 N-terminal amino acids fails to be translocated into peroxisomes but assembles into an enzymatically active form in the cytoplasm of a strain with a disrupted nuclear Thiolase gene. We now report that when truncated Thiolase is cosynthesized with full-length Thiolase, approximately 50% of truncated Thiolase cofractionates with the full-length Thiolase to fractions enriched for peroxisomes and is translocated into peroxisomes as shown by its protection from the action of external proteases. We constructed an immunologically distinct cytosolic variant of Thiolase by adding an influenza hemagglutinin epitope tag to the N terminus of the truncated Thiolase. In a strain simultaneously expressing the full-length, truncated, and epitope-tagged truncated Thiolases, we demonstrated that normally untargeted Thiolase subunits are efficiently translocated into peroxisomes by dimerization with full-length Thiolase subunits. Even though truncated and epitope-tagged truncated Thiolase subunits are translocated into peroxisomes in this strain, only the full-length Thiolase subunit can be coimmunoprecipitated with the epitope-tagged truncated Thiolase subunit from the peroxisomal matrix. This observation suggests that interactions between Thiolase subunits are not disrupted during translocation.
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mutagenesis of the amino targeting signal of saccharomyces cerevisiae 3 ketoacyl coa Thiolase reveals conserved amino acids required for import into peroxisomes in vivo
Journal of Biological Chemistry, 1994Co-Authors: J R Glover, Davidw . Andrews, Suresh Subramani, Richard A RachubinskiAbstract:Abstract Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA Thiolase is a soluble matrix protein that does not end in a consensus peroxisomal targeting signal-1. The amino terminus of S. cerevisiae peroxisomal Thiolase is conserved in 6 of 11 residues with the amino terminus of rat Thiolase B, shown to act as a peroxisomal targeting signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991) EMBO J. 10, 3255-3262). Unlike mammalian peroxisomal Thiolases, there is no extensive cleavage of S. cerevisiae Thiolase upon import into peroxisomes. We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S. cerevisiae Thiolase are necessary and sufficient for targeting to peroxisomes. This result implies that yeast, like mammalian cells, can target proteins to the peroxisomal matrix by at least two different routes. We also demonstrate by targeted mutagenesis and in vivo expression of mutated Thiolase genes that three amino acids conserved in the amino termini of all known Thiolases are critical for efficient targeting of S. cerevisiae Thiolase to peroxisomes.
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a novel cleavable peroxisomal targeting signal at the amino terminus of the rat 3 ketoacyl coa Thiolase
The EMBO Journal, 1991Co-Authors: Bart W Swinkels, Richard A Rachubinski, Stephen J Gould, Andrea G Bodnar, Suresh SubramaniAbstract:Abstract Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy-termini. One such protein is the peroxisomal 3-ketoacyl CoA Thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600-4606]. Both rat peroxisomal Thiolases are synthesized as larger precursors with an amino-terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the Thiolases into peroxisomes because expression of an altered cDNA encoding only the mature Thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the Thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino-terminal or internal locations and is distinct from the C-terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.
Atsuo Tanaka - One of the best experts on this subject based on the ideXlab platform.
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genetic evaluation of physiological functions of Thiolase isozymes in the n alkane assimilating yeast candida tropicalis
Journal of Bacteriology, 1998Co-Authors: Naoki Kanayama, Mitsuyoshi Ueda, Haruyuki Atomi, Atsuo TanakaAbstract:The n-alkane-assimilating diploid yeast Candida tropicalis possesses three Thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) Thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA Thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these Thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA Thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA Thiolase and resulted in growth deficiency on n-alkanes (C10 to C13). All Thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA Thiolases, the site-directed mutation leading acetoacetyl-CoA Thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA Thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA Thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA Thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA Thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA Thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA Thiolase and 3-ketoacyl-CoA Thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA Thiolase contributed greatly even to the β-oxidation of a C4 substrate butyrate.
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Molecular evolution of yeast Thiolase isozymes
Journal of Fermentation and Bioengineering, 1994Co-Authors: Naoki Kanayama, Tatsuo Kurihara, Mitsuyoshi Ueda, Haruyuki Atomi, Atsuo TanakaAbstract:Abstract The coexistence of two Thiolase isozymes (acetoacetyl-CoA Thiolase and 3-ketoacyl-CoA Thiolase), essential for the complete degradation of fatty acids, only in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (Kurihara et al., J. Biochem., 106, 474–478, 1989) is unique in eukaryotic cells. As one of the methods of analysis of molecular information from these isozymes, the calculation of the evolutional distance among Thiolases from various organisms suggested that yeast peroxisomal Thiolase isozymes are important enzymes in examining the molecular evolution of the fatty acid metabolic pathway and the biogenesis of peroxisomes.
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peroxisomal acetoacetyl coa Thiolase of an n alkane utilizing yeast candida tropicalis
FEBS Journal, 1992Co-Authors: Tatsuo Kurihara, Mitsuyoshi Ueda, Naoki Kanayama, Jun Kondo, Yutaka Teranishi, Atsuo TanakaAbstract:Two genes encoding acetoacetyl-CoA Thiolase (Thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the λEMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as Thiolase IA and Thiolase IB. The calculated molecular masses were 41 898 Da for Thiolase IA and 41 930 Da for Thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that Thiolase IA and Thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other Thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA Thiolase (65% amino acids of Thiolase IA and 64% of Thiolase IB were identical with this Thiolase). Considering the evolution of Thiolases, the C. tropicalis Thiolases and S. uvarum cytosolic acetoacetyl-CoA Thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of Thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other Thiolases reported hitherto.
Walter Reineke - One of the best experts on this subject based on the ideXlab platform.
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degradation of aromatics and chloroaromatics by pseudomonas sp strain b13 purification and characterization of 3 oxoadipate succinyl coenzyme a coa transferase and 3 oxoadipyl coa Thiolase
Journal of Bacteriology, 2002Co-Authors: Stefan R Kaschabek, Bernd Kuhn, Dagmar Muller, Eberhard Schmidt, Walter ReinekeAbstract:The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 ± 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-Oxoadipyl-CoA Thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 ± 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA Thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA Thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the Thiolase terminus revealed high percentages of identity (70 to 85%) with Thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.
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degradation of aromatics and chloroaromatics by pseudomonas sp strain b13 cloning characterization and analysis of sequences encoding 3 oxoadipate succinyl coenzyme a coa transferase and 3 oxoadipyl coa Thiolase
Journal of Bacteriology, 2002Co-Authors: Markus Gobel, Eberhard Schmidt, Kerstin Kasselcati, Walter ReinekeAbstract:3-Oxoadipate:succinyl-coenzyme A (CoA) transferase and 3-oxoadipyl-CoA Thiolase carry out the ultimate steps in the conversion of benzoate and 3-chlorobenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the 3-oxoadipate pathway. This report describes the characterization of DNA fragments with the overall length of 5.9 kb from Pseudomonas sp. strain B13 that encode these enzymes. DNA sequence analysis revealed five open reading frames (ORFs) plus an incomplete one. ORF1, of unknown function, has a length of 414 bp. ORF2 (catI) encodes a polypeptide of 282 amino acids and starts at nucleotide 813. ORF3 (catJ) encodes a polypeptide of 260 amino acids and begins at nucleotide 1661. CatI and CatJ are the subunits of the 3-oxoadipate:succinyl-CoA transferase, whose activity was demonstrated when both genes were ligated into expression vector pET11a. ORF4, termed catF, codes for a protein of 401 amino acid residues with a predicted mass of 41,678 Da with 3-oxoadipyl-CoA Thiolase activity. The last three ORFs seem to form an operon since they are oriented in the same direction and showed an overlapping of 1 bp between catI and catJ and of 4 bp between catJ and catF. Conserved functional groups important for the catalytic activity of CoA transferases and Thiolases were identified in CatI, CatJ, and CatF. ORF5 (catD) encodes the 3-oxoadipate enol-lactone hydrolase. An incomplete ORF6 of 1,183 bp downstream of ORF5 and oriented in the opposite direction was found. The protein sequence deduced from ORF6 showed a putative AMP-binding domain signature.
Toshiyuki Fukao - One of the best experts on this subject based on the ideXlab platform.
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the peroxisomal zebrafish scp2 Thiolase type 1 is a weak transient dimer as revealed by crystal structures and native mass spectrometry
Biochemical Journal, 2019Co-Authors: Tiila-riikka Kiema, Toshiyuki Fukao, Werner Schmitz, C J Thapa, Mikko Laitaoja, Mirko M Maksimainen, Juha Rouvinen, Janne Janis, Rik K. WierengaAbstract:The SCP2 (sterol carrier protein 2)-Thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the β-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric Thiolases has previously been poorly characterized. High-resolution crystal structures of the zebrafish SCP2-Thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerized monomeric form has also been captured in the obtained crystals. Four loops at the dimer interface adopt very different conformations in the monomeric form. These loops also shape the active site and their structural changes explain why a competent active site is not present in the monomeric form. Native mass spectrometry studies confirm that the zebrafish SCP2-Thiolase (type-1) as well as its human homolog are weak transient dimers in solution. The crystallographic binding studies reveal the mode of binding of CoA and octanoyl-CoA in the active site, highlighting the conserved geometry of the nucleophilic cysteine, the catalytic acid/base cysteine and the two oxyanion holes. The dimer interface of SCP2-Thiolase (type-1) is equally extensive as in other Thiolase dimers; however, it is more polar than any of the corresponding interfaces, which correlates with the notion that the enzyme forms a weak transient dimer. The structure comparison of the monomeric and dimeric forms suggests functional relevance of this property. These comparisons provide also insights into the structural rearrangements that occur when the folded inactive monomers assemble into the mature dimer.
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the crystal structure of human mitochondrial 3 ketoacyl coa Thiolase t1 insight into the reaction mechanism of its Thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, R K Harijan, Toshiyuki Fukao, Malgorzata Strozyk, Stefan E H Alexson, Rik K. WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA Thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative Thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric Thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative Thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic Thiolase activity, which is predicted to be of physiological importance.
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The crystal structure of human mitochondrial 3‐ketoacyl‐CoA Thiolase (T1): insight into the reaction mechanism of its Thiolase and thioesterase activities
Acta Crystallographica Section D-biological Crystallography, 2014Co-Authors: Tiila-riikka Kiema, R K Harijan, Toshiyuki Fukao, Malgorzata Strozyk, Stefan E H Alexson, Rik K. WierengaAbstract:Crystal structures of human mitochondrial 3-ketoacyl-CoA Thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 A resolution. The structures confirm the tetrameric quaternary structure of this degradative Thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric Thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative Thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nβ2–Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic Thiolase activity, which is predicted to be of physiological importance.
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crystallographic and kinetic studies of human mitochondrial acetoacetyl coa Thiolase the importance of potassium and chloride ions for its structure and function
Biochemistry, 2007Co-Authors: Antti M Haapalainen, Toshiyuki Fukao, Gitte Meriläinen, Naomi Kondo, Paivi Pirila, Rik K. WierengaAbstract:Thiolases are CoA-dependent enzymes which catalyze the formation of a carbon-carbon bond in a Claisen condensation step and its reverse reaction via a thiolytic degradation mechanism. Mitochondrial acetoacetyl-coenzyme A (CoA) Thiolase (T2) is important in the pathways for the synthesis and degradation of ketone bodies as well as for the degradation of 2-methylacetoacetyl-CoA. Human T2 deficiency has been identified in more than 60 patients. A unique property of T2 is its activation by potassium ions. High-resolution human T2 crystal structures are reported for the apo form and the CoA complex, with and without a bound potassium ion. The potassium ion is bound near the CoA binding site and the catalytic site. Binding of the potassium ion at this low-affinity binding site causes the rigidification of a CoA binding loop and an active site loop. Unexpectedly, a high-affinity binding site for a chloride ion has also been identified. The chloride ion is copurified, and its binding site is at the dimer interface, near two catalytic loops. A unique property of T2 is its ability to use 2-methyl-branched acetoacetyl-CoA as a substrate, whereas the other structurally characterized Thiolases cannot utilize the 2-methylated compounds. The kinetic measurements show that T2 can degrade acetoacetyl-CoA and 2-methylacetoacetyl-CoA with similar catalytic efficiencies. For both substrates, the turnover numbers increase approximately 3-fold when the potassium ion concentration is increased from 0 to 40 mM KCl. The structural analysis of the active site of T2 indicates that the Phe325-Pro326 dipeptide near the catalytic cavity is responsible for the exclusive 2-methyl-branched substrate specificity.
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high resolution crystal structures of human cytosolic Thiolase ct a comparison of the active sites of human ct bacterial Thiolase and bacterial kas i
Journal of Molecular Biology, 2005Co-Authors: Petri Kursula, Toshiyuki Fukao, Herkko Sikkila, Naomi Kondo, Rik K. WierengaAbstract:Thiolases belong to a superfamily of condensing enzymes that includes also β-ketoacyl acyl carrier protein synthases (KAS enzymes), involved in fatty acid synthesis. Here, we describe the high resolution structure of human cytosolic acetoacetyl-CoA Thiolase (CT), both unliganded (at 2.3 A resolution) and in complex with CoA (at 1.6 A resolution). CT catalyses the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, which is the first reaction of the metabolic pathway leading to the synthesis of cholesterol. CT is a homotetramer of exact 222 symmetry. There is an excess of positively charged residues at the interdimer surface leading towards the CoA-binding pocket, possibly important for the efficient capture of substrates. The geometry of the catalytic site, including the three catalytic residues Cys92, His 353, Cys383, and the two oxyanion holes, is highly conserved between the human and bacterial Zoogloea ramigera Thiolase. In human CT, the first oxyanion hole is formed by Wat38 (stabilised by Asn321) and NE2(His353), and the second by N(Cys92) and N(Gly385). The active site of this superfamily is constructed on top of four active site loops, near Cys92, Asn321, His353, and Cys383, respectively. These loops were used for the superpositioning of CT on the bacterial Thiolase and on the Escherichia coli KAS I. This comparison indicates that the two Thiolase oxyanion holes also exist in KAS I at topologically equivalent positions. Interestingly, the hydrogen bonding interactions at the first oxyanion hole are different in Thiolase and KAS I. In KAS I, the hydrogen bonding partners are two histidine NE2 atoms, instead of a water and a NE2 side-chain atom in Thiolase. The second oxyanion hole is in both structures shaped by corresponding main chain peptide NH-groups. The possible importance of bound water molecules at the catalytic site of Thiolase for the reaction mechanism is discussed.
