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Anping Zeng - One of the best experts on this subject based on the ideXlab platform.
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formaldehyde formation in the Glycine Cleavage System and its use for an aldolase based biosynthesis of 1 3 prodanediol
Journal of Biological Engineering, 2020Co-Authors: Hao Meng, Jie Ren, Anping ZengAbstract:Glycine Cleavage System (GCS) occupies a key position in one-carbon (C1) metabolic pathway and receives great attention for the use of C1 carbons like formate and CO2 via synthetic biology. In this work, we demonstrate that formaldehyde exists as a substantial byproduct of the GCS reaction cycle. Three causes are identified for its formation. First, the principal one is the decomposition of N5,N10-methylene-tetrahydrofolate (5,10-CH2-THF) to form formaldehyde and THF. Increasing the rate of Glycine Cleavage promotes the formation of 5,10-CH2-THF, thereby increasing the formaldehyde release rate. Next, formaldehyde can be produced in the GCS even in the absence of THF. The reason is that T-protein of the GCS can degrade methylamine-loaded H-protein (Hint) to formaldehyde and ammonia, accompanied with the formation of dihydrolipoyl H-protein (Hred), but the reaction rate is less than 0.16% of that in the presence of THF. Increasing T-protein concentration can speed up the release rate of formaldehyde by Hint. Finally, a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF. Based on a formaldehyde-dependent aldolase, we elaborated a Glycine-based one carbon metabolic pathway for the biosynthesis of 1,3-propanediol (1,3-PDO) in vitro. This work provides quantitative data and mechanistic understanding of formaldehyde formation in the GCS and a new biosynthetic pathway of 1,3-PDO.
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quantitative study of h protein lipoylation of the Glycine Cleavage System and a strategy to increase its activity by co expression of lpla
Journal of Biological Engineering, 2019Co-Authors: Xinyi Zhang, Jie Ren, Anping ZengAbstract:Glycine Cleavage System (GCS) plays a key role in one-carbon (C1) metabolism related to the biosynthesis of a number of key intermediates with significance in both biomedicine and biotechnology. Despite extensive studies of the proteins (H, T, P and L) involved and the reaction mechanisms of this important enzyme complex little quantitative data are available. In this work, we have developed a simple HPLC method for direct analysis and quantification of the apo- and lipoylated forms (Hapo and Hlip) of the shuttle protein H, the latter (Hlip) is essential for the function of H protein and determines the activity of GCS. Effects of temperature, concentrations of lipoic acid and Hapo and the expression of H protein on its lipoylation were studied. It is found that Hlip is as low as only 20–30% of the total H protein with lipoic acid concentration in the range of 10–20 μM and at a favorable temperature of 30 °C. Furthermore, Hapo seems to inhibit the overall activity of GCS. We proposed a strategy of co-expressing LplA to improve the lipoylation of H protein and GCS activity. With this strategy the fraction of Hlip was increased, for example, from 30 to 90% at a lipoic acid concentration of 20 μM and GCS activity was increased by more than 2.5 fold. This work lays a quantitative foundation for better understanding and reengineering the GCS System.
Roland Douce - One of the best experts on this subject based on the ideXlab platform.
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Interaction between Glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria
Biochemical Journal, 1994Co-Authors: Fabrice Rebeille, Michel Neuburger, Roland DouceAbstract:The aim of the present work was to further determine how the T-protein of the Glycine-Cleavage System and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the Glycine-Cleavage System was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of Glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.
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Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and Glycine decarboxylase from pea leaf mitochondria.
Biochemical Journal, 1993Co-Authors: V. Besson, Michel Neuburger, Fabrice Rebeille, Roland Douce, Edwin A CossinsAbstract:Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the Glycine-Cleavage System and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the Glycine decarboxylase T-protein.
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isolation characterization and sequence analysis of a cdna clone encoding l protein the dihydrolipoamide dehydrogenase component of the Glycine Cleavage System from pea leaf mitochondria
FEBS Journal, 1992Co-Authors: Jacques Bourguignon, Michel Neuburger, David Macherel, Roland DouceAbstract:L-protein is the dihydrolipoamide dehydrogenase component of the Glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a λ gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the Glycine-Cleavage System recognized proteins with an Mr of about 60 000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northernblot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the Glycine-Cleavage System) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
Shigeo Kure - One of the best experts on this subject based on the ideXlab platform.
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Glycine Cleavage System in neurogenic regions
2014Co-Authors: Prof Z. Molnar, Shigeo Kure, Kanako Kojima, Akiko Ichinohe, Sumiko Mikawa, Takatoshi Ueki, Kazuko Fujiwara, Kazuie Iinuma, Kohji SatoAbstract:hyperGlycinemia, rat, neural stem cell. Ichinohe A et al. 2 The Glycine Cleavage System (GCS) is the essential enzyme complex for degrading Glycine and supplying 5,10-methylenetetrahydrofolate for DNA synthesis. Inherited deficiency of this System causes non-ketotic hyperGlycinemia, characterized by severe neurological symptoms and frequent association of brain malformations. Although high levels of Glycine have been considered to cause the above-mentioned problems, the detailed pathogenesis of this disease is still unknown. Here we show that GCS is abundantly expressed in rat embryonic neural stem/progenitor cells in the neuroepithelium, and this expression is transmitted to the radial glia-astrocyte lineage, with prominence in postnatal neurogenic regions. These data indicate that GCS plays important roles in neurogenesis, and suggest that disturbance of neurogenesis induced by deficiency of GCS may be the main pathogenesis of non-ketotic hyperGlycinemia
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nonketotic hyperGlycinemia pathophysiological studies
Proceedings of the Japan Academy. Series B Physical and biological sciences, 2005Co-Authors: Keiya Tada, Shigeo KureAbstract:Recent study on nonketotic hyperGlycinemia, an inborn error of Glycine metabolism, is reviewed from clinical, metabolic, molecular, and neuropathological points of view. This disorder is caused by an inherited deficiency of the mitochondrial Glycine Cleavage System (GCS), which causes accumulation of Glycine in such body fluids as plasma, cerebrospinal fluid, and urine. There are four disease types: neonatal, infantile, late-onset, and transient types. The genetic backgrounds of the neonatal and infantile types have been largely clarified by a comprehensive mutational screening of genes encoding three components of the GCS, while the molecular pathogenesis of the late-onset and transient types are largely unknown. In the central nervous System of vertebrates, the GCS has been identified in astrocytes and neural stem cells. The GCS in astrocytes is co-localized with N-methyl-D-aspartate receptors, and is thought to maintain the Glycine level around the receptors, while the physiological and pathological roles of the GCS in neural stem cells remains to be elucidated. (Communicated by Tamio YAMAKAWA, M.J.A.)
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novel mutations in the p protein Glycine decarboxylase gene in patients with Glycine encephalopathy non ketotic hyperGlycinemia
Molecular Genetics and Metabolism, 2002Co-Authors: Shigeo Kure, Kanako Kojima, Jennifer R Toone, Derek A Applegarth, Marion B Coultermackie, Payam Sazegar, Akiko IchinoheAbstract:Eight novel mutations were found in the P-protein (Glycine decarboxylase) gene (GLDC) of the Glycine Cleavage System (EC 2.1.1.10) by screening five exons of the gene in patients with Glycine encephalopathy (NKH). The mutations identified were of eight single base changes: a one-base deletion 1054del A, a splice site mutation IVS18-2A-->G and six amino acid substitutions A283P, A313P, P329T, R410K, P700A, and G762R.
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chromosomal localization structure single nucleotide polymorphisms and expression of the human h protein gene of the Glycine Cleavage System gcsh a candidate gene for nonketotic hyperGlycinemia
Journal of Human Genetics, 2001Co-Authors: Shigeo Kure, Kanako Kojima, Takayuki Kudo, Kiyoshi Kanno, Yoko Aoki, Yoichi Suzuki, Toshikatsu Shinka, Yoshiyuki Sakata, Kuniaki Narisawa, Yoichi MatsubaraAbstract:Nonketotic hyperGlycinemia (NKH) is an inborn error of metabolism caused by deficiency in the Glycine Cleavage System (GCS); this System consists of four individual constituents, P-, T-, H-, and L-proteins. Several mutations have been identified in P- and T-protein genes, but not in the H-protein gene (GCSH), despite the presence of case reports of H-protein deficiency. To facilitate the mutational and functional analyses of GCSH, we isolated and characterized a human p1-derived artificial chromosome (PAC) clone encoding GCSH. GCSH spanned 13.5 kb and consisted of five exons. Using the PAC clone as a probe, we mapped GCSH to chromosome 16q24 by fluorescence in situ hybridization. The transcription initiation site was determined by the oligonucleotide-cap method, and potential binding sites for several transcriptional factors were found in the 5′ upstream region. Direct sequencing analysis revealed five single-nucleotide polymorphisms. The expression profiles of P-, T-, and H-protein mRNAs were studied by dot-blot analysis, using total RNA from various human tissues. GCSH was expressed in all 29 tissues examined, while T-protein mRNA was detected in 27 of the 29 tissues. In contrast, the P-protein gene was expressed in a limited number of tissues, such as liver, kidney, brain, pituitary gland, and thyroid gland, suggesting distinct transcriptional regulation of each GCS constituent.
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increased cerebrospinal fluid Glycine a biochemical marker for a leukoencephalopathy with vanishing white matter
Journal of Child Neurology, 1999Co-Authors: M S Van Der Knaap, Shigeo Kure, Ron A Wevers, F J M Gabreels, N M Verhoeven, A H J Van Raaijselten, J JaekenAbstract:Recently, a new disease entity has been defined: the disease of vanishing white matter. This leukoencephalopathy has an autosomal-recessive mode of inheritance. No cause or biochemical marker is known. We studied cerebrospinal fluid amino acids in five patients with the disease and found a consistent, moderate elevation of cerebrospinal fluid Glycine in all. The ratio of cerebrospinal fluid to plasma Glycine was elevated in four patients, in two patients reaching the level considered diagnostic for nonketotic hyperGlycinemia. The activity of the Glycine Cleavage System was found to be normal in lymphoblasts in two patients. The elevation of cerebrospinal fluid Glycine in the disease of vanishing white matter is either caused by a primary disturbance of Glycine metabolism or is secondary to excitotoxic brain damage. (J Child Neurol 1999;14:728-731).
Michel Neuburger - One of the best experts on this subject based on the ideXlab platform.
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Interaction between Glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria
Biochemical Journal, 1994Co-Authors: Fabrice Rebeille, Michel Neuburger, Roland DouceAbstract:The aim of the present work was to further determine how the T-protein of the Glycine-Cleavage System and serine hydroxy-methyltransferase (SHMT), two folate-dependent enzymes from pea leaf mitochondria, interact through a common pool of tetrahydrofolate polyglutamates (H4PteGlun). It was observed that the binding affinity of tetrahydrofolate polyglutamates for these proteins continuously increased with increasing number of glutamates up to six residues. It was also established that, once bound to the proteins, tetrahydrofolate, a very O2-sensitive molecule, was protected from oxidative degradation. The dissociation constants (Kd) of H4PteGlu5, the most predominant form of polyglutamate in the mitochondria, were approximately 0.5 microM for both T-protein and SHMT, whereas the Kd values of CH2-H4PteGlu5 were higher, 2.7 and 7 microM respectively. In a matrix extract from pea leaf mitochondria, the maximal activity of the Glycine-Cleavage System was about 2.5 times higher than the maximal activity of SHMT. This resulted in a permanent disequilibrium of the SHMT-catalysed reaction which was therefore driven toward the production of serine and H4PteGlun, the thermodynamically unfavourable direction. Indeed, measurements of the steady-state ratio of CH2-H4PteGlun/H4PteGlun (n = 1 or n = 5) during the course of Glycine oxidation demonstrated that the methylene form accounted for 65-80% of the folate pool. This indicates that, in our in vitro experiments, CH2-H4PteGlun with long polyglutamate chains accumulated in the bulk medium. This observation suggests that, in these in vitro experiments at least, there was no channelling of CH2-H4PteGlu5 between the T-protein and SHMT.
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Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyltransferase and Glycine decarboxylase from pea leaf mitochondria.
Biochemical Journal, 1993Co-Authors: V. Besson, Michel Neuburger, Fabrice Rebeille, Roland Douce, Edwin A CossinsAbstract:Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the Glycine-Cleavage System and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the Glycine decarboxylase T-protein.
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isolation characterization and sequence analysis of a cdna clone encoding l protein the dihydrolipoamide dehydrogenase component of the Glycine Cleavage System from pea leaf mitochondria
FEBS Journal, 1992Co-Authors: Jacques Bourguignon, Michel Neuburger, David Macherel, Roland DouceAbstract:L-protein is the dihydrolipoamide dehydrogenase component of the Glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a λ gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the Glycine-Cleavage System recognized proteins with an Mr of about 60 000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northernblot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the Glycine-Cleavage System) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.
Yutaro Motokawa - One of the best experts on this subject based on the ideXlab platform.
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Glycine Cleavage System reaction mechanism physiological significance and hyperGlycinemia
Proceedings of the Japan Academy. Series B Physical and biological sciences, 2008Co-Authors: Goro Kikuchi, Yutaro Motokawa, Tadashi Yoshida, Koichi HiragaAbstract:The Glycine Cleavage System catalyzes the following reversible reaction: Glycine + H(4)folate + NAD(+) 5,10-methylene-H(4)folate + CO(2) + NH(3) + NADH + H(+)The Glycine Cleavage System is widely distributed in animals, plants and bacteria and consists of three intrinsic and one common components: those are i) P-protein, a pyridoxal phosphate-containing protein, ii) T-protein, a protein required for the tetrahydrofolate-dependent reaction, iii) H-protein, a protein that carries the aminomethyl intermediate and then hydrogen through the prosthetic lipoyl moiety, and iv) L-protein, a common lipoamide dehydrogenase. In animals and plants, the proteins form an enzyme complex loosely associating with the mitochondrial inner membrane. In the enzymatic reaction, H-protein converts P-protein, which is by itself a potential alpha-amino acid decarboxylase, to an active enzyme, and also forms a complex with T-protein. In both Glycine Cleavage and synthesis, aminomethyl moiety bound to lipoic acid of H-protein represents the intermediate that is degraded to or can be formed from N(5),N(10)-methylene-H(4)folate and ammonia by the action of T-protein. N(5),N(10)-Methylene-H(4)folate is used for the biosynthesis of various cellular substances such as purines, thymidylate and methionine that is the major methyl group donor through S-adenosyl-methionine. This accounts for the physiological importance of the Glycine Cleavage System as the most prominent pathway in serine and Glycine catabolism in various vertebrates including humans. Nonketotic hyperGlycinemia, a congenital metabolic disorder in human infants, results from defective Glycine Cleavage activity. The majority of patients with nonketotic hyperGlycinemia had lesions in the P-protein gene, whereas some had mutant T-protein genes. The only patient classified into the degenerative type of nonketotic hyperGlycinemia had an H-protein devoid of the prosthetic lipoyl residue. The crystallography of normal T-protein as well as biochemical characterization of recombinants of the normal and mutant T-proteins confirmed why the mutant T-proteins had lost enzyme activity. Putative mechanisms of cellular injuries including those in the central nervous System of patients with nonketotic hyperGlycinemia are discussed.
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crystal structure of human t protein of Glycine Cleavage System at 2 0 a resolution and its implication for understanding non ketotic hyperGlycinemia
Journal of Molecular Biology, 2005Co-Authors: K Okamuraikeda, Yutaro Motokawa, Kazuko Fujiwara, Harumi Hosaka, M Yoshimura, Eiki Yamashita, Sachiko Toma, Atsushi Nakagawa, Hisaaki TaniguchiAbstract:T-protein, a component of the Glycine Cleavage System, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of Glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperGlycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH 3 -H 4 folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH 3 -H 4 folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of α-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH 3 -H 4 folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.
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identification of the folate binding sites on the escherichia coli t protein of the Glycine Cleavage System
Journal of Biological Chemistry, 1999Co-Authors: K Okamuraikeda, Kazuko Fujiwara, Yutaro MotokawaAbstract:Abstract T-protein is a component of the Glycine Cleavage System and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme,14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5,10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5,10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) inK m values for both folate substrates, but that of Lys-81 affected greatly the K m value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to α-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to α-carboxyl group of the second glutamate residue.
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expression of mature bovine h protein of the Glycine Cleavage System in escherichia coli and in vitro lipoylation of the apoform
Journal of Biological Chemistry, 1992Co-Authors: Kazuko Fujiwara, K Okamuraikeda, Yutaro MotokawaAbstract:H-protein, a component of the Glycine Cleavage System with lipoic acid as a prosthetic group, was expressed in Escherichia coli using a T7 RNA polymerase plasmid expression System. After induction with 25 microM isopropyl-beta-D-thiogalactopyranoside, bacteria harboring the recombinant plasmid expressed mature bovine H-protein as a soluble form at a level of about 10% of the total bacterial protein. Little of the H-protein was lipoylated in E. coli cultured without added lipoate, but when the cells were cultured in medium supplemented with 30 microM lipoate, about 10% of the recombinant protein expressed was the correctly lipoylated active form, 10% was an inactive aberrantly modified form, presumably with an octanoyl group, and the remaining 80% was the unlipoylated apoform. Each of the three forms was purified to homogeneity and shown to have the same NH2-terminal amino acid sequence as that of native bovine H-protein. The specific activity of the lipoylated form of H-protein expressed was consistent with that of H-protein purified from bovine liver. The purified recombinant apo-H-protein was lipoylated and consequently activated in vitro with lipoyl-AMP as a lipoyl donor by lipoyltransferase purified 150-fold from bovine liver mitochondria. The lipoylation was dependent on lipoyl-AMP, apo-H-protein, and lipoyltransferase. The partially purified lipoyltransferase had no lipoate-activating activity. These results provide the first evidence that in mammals two consecutive reactions are required for the attachment of lipoic acid to the acceptor protein: the activation of lipoic acid to lipoyl-AMP catalyzed by lipoate-activating enzyme and the transfer of the lipoyl group to an N epsilon-amino group of a lysine residue to apoprotein by lipoyl-AMP:N epsilon-lysine lipoyltransferase.