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Christopher J Schofield - One of the best experts on this subject based on the ideXlab platform.
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synthesis of 2 oxoglutarate derivatives and their evaluation as cosubstrates and inhibitors of human aspartate asparagine β hydroxylase
Chemical Science, 2021Co-Authors: Lennart Brewitz, Yu Nakashima, Christopher J SchofieldAbstract:2-Oxoglutarate (2OG) is involved in biological processes including oxidations catalyzed by 2OG Oxygenases for which it is a cosubstrate. Eukaryotic 2OG Oxygenases have roles in collagen biosynthesis, lipid metabolism, DNA/RNA modification, transcriptional regulation, and the hypoxic response. Aspartate/asparagine-β-hydroxylase (AspH) is a human 2OG Oxygenase catalyzing post-translational hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs) in the endoplasmic reticulum. AspH is of chemical interest, because its Fe(II) cofactor is complexed by two rather than the typical three residues. AspH is upregulated in hypoxia and is a prognostic marker on the surface of cancer cells. We describe studies on how derivatives of its natural 2OG cosubstrate modulate AspH activity. An efficient synthesis of C3- and/or C4-substituted 2OG derivatives, proceeding via cyanosulfur ylid intermediates, is reported. Mass spectrometry-based AspH assays with >30 2OG derivatives reveal that some efficiently inhibit AspH via competing with 2OG as evidenced by crystallographic and solution analyses. Other 2OG derivatives can substitute for 2OG enabling substrate hydroxylation. The results show that subtle changes, e.g. methyl- to ethyl-substitution, can significantly alter the balance between catalysis and inhibition. 3-Methyl-2OG, a natural product present in human nutrition, was the most efficient alternative cosubstrate identified; crystallographic analyses reveal the binding mode of (R)-3-methyl-2OG and other 2OG derivatives to AspH and inform on the balance between turnover and inhibition. The results will enable the use of 2OG derivatives as mechanistic probes for other 2OG utilizing enzymes and suggest 2-oxoacids other than 2OG may be employed by some 2OG Oxygenases in vivo.
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kinetic parameters of human aspartate asparagine β hydroxylase suggest that it has a possible function in oxygen sensing
Journal of Biological Chemistry, 2020Co-Authors: Lennart Brewitz, Anthony Tumber, Christopher J SchofieldAbstract:Human aspartate/asparagine-β-hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent Oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor-like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1-2, 3-4, 5-6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG Oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG Oxygenases.
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Structure of Human Phytanoyl-CoA 2-Hydroxylase Identifies Molecular Mechanisms of Refsum Disease
The Journal of biological chemistry, 2005Co-Authors: Michael A. Mcdonough, Timothy Searls, Danica Butler, Kathryn L. Kavanagh, Udo Oppermann, Christopher J SchofieldAbstract:Abstract Refsum disease (RD), a neurological syndrome characterized by adult onset retinitis pigmentosa, anosmia, sensory neuropathy, and phytanic acidaemia, is caused by elevated levels of phytanic acid. Many cases of RD are associated with mutations in phytanoyl-CoA 2-hydroxylase (PAHX), an Fe(II) and 2-oxoglutarate (2OG)-dependent Oxygenase that catalyzes the initial α-oxidation step in the degradation of phytenic acid in peroxisomes. We describe the x-ray crystallographic structure of PAHX to 2.5 A resolution complexed with Fe(II) and 2OG and predict the molecular consequences of mutations causing RD. Like other 2OG Oxygenases, PAHX possesses a double-stranded β-helix core, which supports three iron binding ligands (His175, Asp177, and His264); the 2-oxoacid group of 2OG binds to the Fe(II) in a bidentate manner. The manner in which PAHX binds to Fe(II) and 2OG together with the presence of a cysteine residue (Cys191) 6.7 A from the Fe(II) and two further histidine residues (His155 and His281) at its active site distinguishes it from that of the other human 2OG Oxygenase for which structures are available, factor inhibiting hypoxia-inducible factor. Of the 15 PAHX residues observed to be mutated in RD patients, 11 cluster in two distinct groups around the Fe(II) (Pro173, His175, Gln176, Asp177, and His220) and 2OG binding sites (Trp193, Glu197, Ile199, Gly204, Asn269, and Arg275). PAHX may be the first of a new subfamily of coenzyme A-binding 2OG Oxygenases.
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structure and mechanism of anthocyanidin synthase from arabidopsis thaliana
Structure, 2002Co-Authors: Rupert C Wilmouth, Ian J Clifton, Richard W D Welford, Jonathan J Turnbull, Andrea G Prescott, Christopher J SchofieldAbstract:Flavonoids are common colorants in plants and have long-established biomedicinal properties. Anthocyanidin synthase (ANS), a 2-oxoglutarate iron-dependent Oxygenase, catalyzes the penultimate step in the biosynthesis of the anthocyanin class of flavonoids. The crystal structure of ANS reveals a multicomponent active site containing metal, cosubstrate, and two molecules of a substrate analog (dihydroquercetin). An additional structure obtained after 30 min exposure to dioxygen is consistent with the oxidation of the dihydroquercetin to quercetin and the concomitant decarboxylation of 2-oxoglutarate to succinate. Together with in vitro studies, the crystal structures suggest a mechanism for ANS-catalyzed anthocyanidin formation from the natural leucoanthocyanidin substrates involving stereoselective C-3 hydroxylation. The structure of ANS provides a template for the ubiquitous family of plant nonhaem Oxygenases for future engineering and inhibition studies.
Takayuki Kohchi - One of the best experts on this subject based on the ideXlab platform.
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The Arabidopsis Photomorphogenic Mutant hy1 Is Deficient in Phytochrome Chromophore Biosynthesis as a Result of a
2013Co-Authors: Mutation Plastid, Takuya Muramoto, Akiho Yokota, Takayuki Kohchi, Heme Oxygenase, Inhwan A Hwang, Howard Goodman M. BAbstract:The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme Oxygenases and contains a possible transit peptide for transport to plastids. Heme Oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme Oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme Oxygenase necessary for phytochrome chromophore biosynthesis
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expression and biochemical properties of a ferredoxin dependent heme Oxygenase required for phytochrome chromophore synthesis
Plant Physiology, 2002Co-Authors: Takuya Muramoto, Noriyuki Tsurui, Matthew J Terry, Akiho Yokota, Takayuki KohchiAbstract:The HY1 gene of Arabidopsis encodes a plastid heme Oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme Oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXα from heme with the concomitant production of carbon monoxide. Heme Oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme Oxygenases differ substantially.
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expression and biochemical properties of a ferredoxin dependent heme Oxygenase required for phytochrome chromophore synthesis
Plant Physiology, 2002Co-Authors: Takuya Muramoto, Noriyuki Tsurui, Matthew J Terry, Akiho Yokota, Takayuki KohchiAbstract:The HY1 gene of Arabidopsis encodes a plastid heme Oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme Oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXα from heme with the concomitant production of carbon monoxide. Heme Oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme Oxygenases differ substantially.
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the arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme Oxygenase
The Plant Cell, 1999Co-Authors: Takuya Muramoto, Akiho Yokota, Takayuki Kohchi, Inhwan Hwang, Howard M GoodmanAbstract:The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme Oxygenases and contains a possible transit peptide for transport to plastids. Heme Oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme Oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme Oxygenase necessary for phytochrome chromophore biosynthesis.
Martin Munzel - One of the best experts on this subject based on the ideXlab platform.
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aspartate asparagine β hydroxylase crystal structures reveal an unexpected epidermal growth factor like domain substrate disulfide pattern
Nature Communications, 2019Co-Authors: Inga Pfeffer, Ks Hewitson, Nadia J. Kershaw, Luke A. Mcneill, T Krojer, Lennart Brewitz, Sacha A Jensen, Grazyna Kochan, Holger B Kramer, Martin MunzelAbstract:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate Oxygenase whose C-terminal Oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the Oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG Oxygenases.
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aspartate asparagine β hydroxylase crystal structures reveal an unexpected epidermal growth factor like domain substrate disulfide pattern
Nature Communications, 2019Co-Authors: Inga Pfeffer, Ks Hewitson, Nadia J. Kershaw, Luke A. Mcneill, T Krojer, Lennart Brewitz, Sacha A Jensen, Grazyna Kochan, Holger B Kramer, Martin MunzelAbstract:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate Oxygenase whose C-terminal Oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the Oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG Oxygenases. AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.
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ycfdrm is a thermophilic oxygen dependent ribosomal protein ul16 Oxygenase
Extremophiles, 2018Co-Authors: Rok Sekirnik, Sarah E Wilkins, Hanna Tarhonskaya, Jacob T Bush, Emily Flashman, Martin Munzel, Aayan Hussein, Shabaz Mohammed, Michael A. McdonoughAbstract:YcfD from Escherichia coli is a homologue of the human ribosomal Oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960–962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG Oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG Oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG Oxygenases are of interest from a biocatalytic perspective.
Lennart Brewitz - One of the best experts on this subject based on the ideXlab platform.
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synthesis of 2 oxoglutarate derivatives and their evaluation as cosubstrates and inhibitors of human aspartate asparagine β hydroxylase
Chemical Science, 2021Co-Authors: Lennart Brewitz, Yu Nakashima, Christopher J SchofieldAbstract:2-Oxoglutarate (2OG) is involved in biological processes including oxidations catalyzed by 2OG Oxygenases for which it is a cosubstrate. Eukaryotic 2OG Oxygenases have roles in collagen biosynthesis, lipid metabolism, DNA/RNA modification, transcriptional regulation, and the hypoxic response. Aspartate/asparagine-β-hydroxylase (AspH) is a human 2OG Oxygenase catalyzing post-translational hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs) in the endoplasmic reticulum. AspH is of chemical interest, because its Fe(II) cofactor is complexed by two rather than the typical three residues. AspH is upregulated in hypoxia and is a prognostic marker on the surface of cancer cells. We describe studies on how derivatives of its natural 2OG cosubstrate modulate AspH activity. An efficient synthesis of C3- and/or C4-substituted 2OG derivatives, proceeding via cyanosulfur ylid intermediates, is reported. Mass spectrometry-based AspH assays with >30 2OG derivatives reveal that some efficiently inhibit AspH via competing with 2OG as evidenced by crystallographic and solution analyses. Other 2OG derivatives can substitute for 2OG enabling substrate hydroxylation. The results show that subtle changes, e.g. methyl- to ethyl-substitution, can significantly alter the balance between catalysis and inhibition. 3-Methyl-2OG, a natural product present in human nutrition, was the most efficient alternative cosubstrate identified; crystallographic analyses reveal the binding mode of (R)-3-methyl-2OG and other 2OG derivatives to AspH and inform on the balance between turnover and inhibition. The results will enable the use of 2OG derivatives as mechanistic probes for other 2OG utilizing enzymes and suggest 2-oxoacids other than 2OG may be employed by some 2OG Oxygenases in vivo.
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kinetic parameters of human aspartate asparagine β hydroxylase suggest that it has a possible function in oxygen sensing
Journal of Biological Chemistry, 2020Co-Authors: Lennart Brewitz, Anthony Tumber, Christopher J SchofieldAbstract:Human aspartate/asparagine-β-hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent Oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor-like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1-2, 3-4, 5-6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG Oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG Oxygenases.
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aspartate asparagine β hydroxylase crystal structures reveal an unexpected epidermal growth factor like domain substrate disulfide pattern
Nature Communications, 2019Co-Authors: Inga Pfeffer, Ks Hewitson, Nadia J. Kershaw, Luke A. Mcneill, T Krojer, Lennart Brewitz, Sacha A Jensen, Grazyna Kochan, Holger B Kramer, Martin MunzelAbstract:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate Oxygenase whose C-terminal Oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the Oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG Oxygenases.
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aspartate asparagine β hydroxylase crystal structures reveal an unexpected epidermal growth factor like domain substrate disulfide pattern
Nature Communications, 2019Co-Authors: Inga Pfeffer, Ks Hewitson, Nadia J. Kershaw, Luke A. Mcneill, T Krojer, Lennart Brewitz, Sacha A Jensen, Grazyna Kochan, Holger B Kramer, Martin MunzelAbstract:AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate Oxygenase whose C-terminal Oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the Oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG Oxygenases. AspH catalyses hydroxylation of asparagine and aspartate residues in epidermal growth factor-like domains (EGFDs). Here, the authors present crystal structures of AspH with and without substrates and show that AspH uses EFGD substrates with a non-canonical disulfide pattern.
Takuya Muramoto - One of the best experts on this subject based on the ideXlab platform.
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The Arabidopsis Photomorphogenic Mutant hy1 Is Deficient in Phytochrome Chromophore Biosynthesis as a Result of a
2013Co-Authors: Mutation Plastid, Takuya Muramoto, Akiho Yokota, Takayuki Kohchi, Heme Oxygenase, Inhwan A Hwang, Howard Goodman M. BAbstract:The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme Oxygenases and contains a possible transit peptide for transport to plastids. Heme Oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme Oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme Oxygenase necessary for phytochrome chromophore biosynthesis
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expression and biochemical properties of a ferredoxin dependent heme Oxygenase required for phytochrome chromophore synthesis
Plant Physiology, 2002Co-Authors: Takuya Muramoto, Noriyuki Tsurui, Matthew J Terry, Akiho Yokota, Takayuki KohchiAbstract:The HY1 gene of Arabidopsis encodes a plastid heme Oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme Oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXα from heme with the concomitant production of carbon monoxide. Heme Oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme Oxygenases differ substantially.
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expression and biochemical properties of a ferredoxin dependent heme Oxygenase required for phytochrome chromophore synthesis
Plant Physiology, 2002Co-Authors: Takuya Muramoto, Noriyuki Tsurui, Matthew J Terry, Akiho Yokota, Takayuki KohchiAbstract:The HY1 gene of Arabidopsis encodes a plastid heme Oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme Oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXα from heme with the concomitant production of carbon monoxide. Heme Oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme Oxygenases differ substantially.
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the arabidopsis photomorphogenic mutant hy1 is deficient in phytochrome chromophore biosynthesis as a result of a mutation in a plastid heme Oxygenase
The Plant Cell, 1999Co-Authors: Takuya Muramoto, Akiho Yokota, Takayuki Kohchi, Inhwan Hwang, Howard M GoodmanAbstract:The HY1 locus of Arabidopsis is necessary for phytochrome chromophore biosynthesis and is defined by mutants that show a long hypocotyl phenotype when grown in the light. We describe here the molecular cloning of the HY1 gene by using chromosome walking and mutant complementation. The product of the HY1 gene shows significant similarity to animal heme Oxygenases and contains a possible transit peptide for transport to plastids. Heme Oxygenase activity was detected in the HY1 protein expressed in Escherichia coli. Heme Oxygenase catalyzes the oxygenation of heme to biliverdin, an activity that is necessary for phytochrome chromophore biosynthesis. The predicted transit peptide is sufficient to transport the green fluorescent protein into chloroplasts. The accumulation of the HY1 protein in plastids was detected by using immunoblot analysis with an anti-HY1 antiserum. These results indicate that the Arabidopsis HY1 gene encodes a plastid heme Oxygenase necessary for phytochrome chromophore biosynthesis.
