Decarboxylation of glycine by serine hydroxymethyltransferase in the presence of lipoic acid

Serine hydroxymethyltransferase and the glycine cleavage system are both present in liver mitochondria and both bind glycine to form a pyridoxal 5'-phosphate carbanionic quinoid species. Lipoic acid has been shown to have the ability to intercept the carbanionic intermediate formed from the binary complex of serine hydroxymethyltransferase and glycine and form an intermediate adduct which is ultimately processed to yield CO2 and a methylamine adduct. Kinetic studies have shown that the lipoic acid-dependent decarboxylation of glycine catalyzed by serine hydroxymethyltransferase proceeds through a sequential mechanism. This lipoic acid-dependent decarboxylation catalyzed by serine hydroxymethyltransferase is similar to the initial reaction of the glycine cleavage system and to the lipoic acid-dependent decarboxylation of glycine by the P-protein alone suggesting that both enzymes could serve in lieu of each other.     

The mitochondrial glycine cleavage system

Summary The glycine cleavage enzyme system is composed of four different proteins tentatively called P-protein, H-protein, T-protein and L-protein, and catalyzes the following reaction reversibly: Glycine + tetrahydrofolate + NAD⁺ 5, 10-methylene-tetrahydrofolate + NH₃ + CO₂ + NADH + H^– Glycine decarboxylase, tentatively called P-protein, is able by itself to catalyze glycine decarboxylation, yielding methylamine as product, but at an extremely low rate. P-Protein alone is also able to catalyze slightly the exchange of carboxyl carbon of glycine with CO₂. However, the rates of the P-protein-catalyzed reactions are greatly increased by the co-existence of aminomethyl carrier protein, a lipoic acid-containing enzyme tentatively called H-protein. Several lines of evidence suggest that H-protein brings about a conformational change of P-protein which may be relevant to the expression of the decarboxylase activity of P-protein and that the functional glycine decarboxylase may be an enzyme complex composed of both P-protein and H-protein. H-Protein seems to play a dual role in the glycine decarboxylation; the one as a regulatory protein of P-protein, and the other as an electron-pulling agent and concomitantly as a carrier of the aminomethyl moiety derived from glycine. The idea that H-protein functions as a modulator of P-protein was further supported by the study of a patient with nonketotic hyperglycinemia. The primary lesion in this patient appeared to consist in structural abnormality in H-protein; the H-protein purified from the liver of this patient was apparently devoid of functional lipoic acid. Nevertheless, H-protein from the patient could stimulate the P-protein-catalyzed exchange of the carboxyl carbon of glycine and CO₂, although only to a limited extent. The observed activity should be independent of the functioning of lipoic acid and would be a reflection of a conformational change in P-protein brought about by H-protein.P-Protein was inactivated when it was incubated with glycine in the presence of II-protein, and the inactivation was completely prevented when bicarbonate was further added so as to allow the glycine-CO₂ exchange to proceed. The inactivation was accompanied by a spectral change of P-protein. The inactivation of P-protein seemed to take place as a side reaction of the glycine decarboxylation and to reflect the formation of a ternary complex of P-protein, H-protein and aminomethyl moiety of glycine through a Schiff base linkage of the H-protein-bound aminomethyl moiety with the pyridoxal phosphate of P-protein.     

Hydrogen carrier protein from chicken liver: purification, characterization, and role of its prosthetic group, lipolic acid, in the glycine cleavage reaction.

Hydrogen carrier protein (H-protein), a component of the glycine cleavage system, has been purified to homogeneity from chicken liver mitochondria. The molecular weight and the partial specific volume determined by two different methods were 14,500 and 0.724 ml/g, respectively. The protein has an isoelectric point of 4.0. Amino acid analysis revealed 131 residues, about one-third of which are acidic residues. Evidence is presented indicating that the protein contains one lipoic acid moiety per molecule. In the decarboxylation of glycine the disulfide of the lipoyl moiety is cleaved and one of the resultant sulf-hydryl groups receives an intermediate derived from glycine.     

The glycine decarboxylase system: a fascinating complex

The mitochondrial glycine decarboxylase multienzyme system, connected to serine hydroxymethyltransferase through a soluble pool of tetrahydrofolate, consists of four different component enzymes, the P-, H-, T- and L-proteins. In a multi-step reaction, it catalyses the rapid destruction of glycine molecules flooding out of the peroxisomes during the course of photorespiration. In green leaves, this multienzyme system is present at tremendously high concentrations within the mitochondrial matrix. The structure, mechanism and biogenesis of glycine decarboxylase are discussed. In the catalytic cycle of glycine decarboxylase, emphasis is given to the lipoate-dependent H-protein that plays a pivotal role, acting as a mobile substrate that commutes successively between the other three proteins. Plant mitochondria possess all the necessary enzymatic equipment for de novo synthesis of tetrahydrofolate and lipoic acid, serving as cofactors for glycine decarboxylase and serine hydroxymethyltransferase functioning.     

Mechanism of the glycine cleavage reaction. Properties of the reverse reaction catalyzed by T-protein

T-protein, one of the components of the glycine cleavage system, catalyzes the synthesis of the H-protein-bound intermediate from methylenetetrahydrofolate, ammonia, and H-protein having a reduced lipoyl prosthetic group (Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J. Biol. Chem. 257, 135-139). Spectroscopic studies indicated that the utilization of methylenetetrahydrofolate occurred only in the presence of the three substrates, indicating the formation of a quaternary complex. The amount of methylenetetrahydrofolate consumed was equal to that of methylene carbon attached to H-protein. Steady-state kinetic studies show that the reaction proceeds through an Ordered Ter Bi mechanism. Reduced H-protein is the first substrate that binds T-protein followed by methylenetetrahydrofolate and ammonia. The order of release of products is tetrahydrofolate and the H-protein-bound intermediate. Km values for H-protein, methylenetetrahydrofolate, and ammonia are 0.55 microM, 0.32 mM, and 22 mM, respectively.     

The mechanism of dextransucrase action. Direction of dextran biosynthesis

Appropriate combinations of purified components of the reversible glycine cleavage system of rat liver catalyze three partial reactions: (1) decarboxylation of glycine or its reverse reaction catalyzed by P- and H-protein, (2) condensation of one carbon substrate and ammonia or its reverse reaction catalyzed by T- and H-protein, and (3) oxidation and reduction of active disulfide of H-protein catalyzed by L-protein. Reactions (1) and (2) give the same product which is bound to H-protein. The protein-bound product was isolated by gel filtration and converted to glycine by incubation with P-protein and CO₂ or degraded further to one carbon unit and ammonia by incubation with T-protein and tetrahydrofolate. The data are consistent with the conclusion that the enzyme-bound product is an intermediate in the reversible glycine cleavage reaction. A scheme is presented for the reactions catalyzed by the enzyme system.     

Expression of mature bovine H-protein of the glycine cleavage system in Escherichia coli and in vitro lipoylation of the apoform.

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.     

Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase

A cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with a K(0.5) of 30 microm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria.     

Chicken liver H-protein, a component of the glycine cleavage system. Amino acid sequence and identification of the N epsilon-lipoyllysine residue

The complete amino acid sequence of H-protein from chicken liver was determined by aligning peptides obtained by cyanogen bromide, endoproteinase Lys-C, Staphylococcus aureus V8 protease, and chymotrypsin cleavage together with the partial NH2- and COOH-terminal sequence of the intact protein. H-protein consists of 125 amino acids and a lipoic acid moiety linked to lysine 59. The sequence is: (sequence in text). The lysyl residue involved in lipoic acid attachment is indicated with an asterisk. The molecular weight including lipoic acid is calculated to be 13,883. From the secondary structure predicted by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1978) Adv. Enzymol. 47, 45-148) the lipoic acid binding region shows alpha-helical structure and is predicted to be an interior portion of the protein from the hydropathic profile according to Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132).     

Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid

Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.     

Crystal structure of T-protein of the glycine cleavage system. Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia

The glycine cleavage system catalyzes the oxidative decarboxylation of glycine in bacteria and in mitochondria of animals and plants. Its deficiency in human causes nonketotic hyperglycinemia, an inborn error of glycine metabolism. T-protein, one of the four components of the glycine cleavage system,is a tetrahydrofolate dependent aminomethyltransferase. It catalyzes the transfer of the methylene carbon unit to tetrahydrofolate from the methylamine group covalently attached to the lipoamide arm of H-protein. To gain insight into the T-protein function at the molecular level, we have determined the first crystal structure of T-protein from Thermotoga maritima by the multiwavelength anomalous diffraction method of x-ray crystallography and refined four structures: the apoform; the tetrahydrofolate complex; the folinic acid complex; and the lipoic acid complex. The overall fold of T-protein is similar to that of the C-terminal tetrahydrofolate-binding region (residues 421-830) of Arthrobacter globiformis dimethylglycine oxidase. Tetrahydrofolate (or folinic acid) is bound near the center of the tripartite T-protein. Lipoic acid is bound adjacent to the tetrahydrofolate binding pocket, thus defining the interaction surface for H-protein binding. A homology model of the human T-protein provides the structural framework for understanding the molecular mechanisms underlying the development of nonketotic hyperglycinemia due to missense mutations of the human T-protein.     

The mitochondrial glycine cleavage system: differential inhibition by divalent cations of glycine synthesis and glycine decarboxylation in the glycine-CO2 exchange

The exchange of glycine carboxyl carbon with CO2 catalyzed by the combination of chicken liver glycine decarboxylase (P-protein) and aminomethyl carrier protein (H-protein) was markedly inhibited by various divalent cations, although extents of inhibition by individual metal ions varied considerably. Cu2+ and Zn2+, at 100 microM, inhibited the reaction almost completely, and the inhibitions by Co2+ and Ni2+ were also significant, while Mg2+ and Mn2+ did not appreciably affect the reaction. The inhibition by Zn2+ was competitive with both bicarbonate and H-protein and non-competitive with glycine. Of the two reactions involved in the glycine-CO2 exchange, decarboxylation of glycine yielding the H-protein-bound aminomethyl moiety was not significantly affected by 100 microM Zn2+ or Cu2+, but carboxylation of the H-protein-bound aminomethyl moiety to form glycine was strongly inhibited by either Zn2+ or Cu2+. Various degrees of inhibition of the glycine-CO2 exchange by other divalent metal ions could also be accounted for by the inhibition of the carboxylation step of the exchange reaction. The primary site of the action of divalent metal ions is likely to be not P-protein but H-protein, and the binding of metal ions with the H-protein-bound intermediate of glycine decarboxylation was assumed to account for the observed marked inhibition.     

Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Inhibition of the conversion of glycine to serine in spinach leaf mitochondria

Isonicotinyl hydrazide, glycine hydroxamate, aminoacetonitrile and KCN inhibited the conversion of glycine to serine in spinach ( Spinacea oleracea L. cv. Viking II) mitochondria. The site of inhibition for the different inhibitors was studied. Isonicotinyl hydrazide and glycine hydroxamate both inhibited the partial reactions glycine-bicarbonate exchange and serine hydroxymethyltransferase. The inhibition was competitive for the exchange reaction and noncompetitive for serine hydroxymethyltransferase. Aminoacetonitrile at low concentration (1 m M ) inhibited the glycine-bicarbonate exchange specifically, whereas serine hydroxymethyltransferase was inhibited only at higher concentrations. Aminoacetonitrile was a competitive inhibitor for both reactions. The serine hydroxymethyltransferase was inhibited by KCN whereas the glycine-bicarbonate exchange was only partially inhibited. The KCN-inhibition of serine hydroxymethyltransferase was competitive.     

Inactivation of D-(-)-beta-hydroxybutyrate dehydrogenase by modifiers of carboxyl and histidyl groups

Data presented in this paper suggest that D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains an essential carboxyl group and an essential histidyl residue at or near the active site. Lactate and malate dehydrogenases, which catalyze reactions analogous to that catalyzed by BDH, also contain an aspartyl and a histidyl residue at the active site [Birktoft, J.J., & Banaszak, L.J. (1983) J. Biol. Chem. 258, 472-482]. In addition, all three enzymes contain an essential arginyl residue, apparently concerned with electrostatic interaction with their respective carboxylic acid substrates, and promote ternary adduct formation involving the enzyme, NAD, and sulfite.     

An Escherichia coli protein with homology to the H-protein of the glycine cleavage enzyme complex from pea and chicken liver.

The nucleotide sequence of an Escherichia coli gene which presumably encodes the H-protein of the glycine cleavage (GCV) enzyme complex is presented. The gene, designated gcvH, encodes a polypeptide of 128 amino acids with a calculated molecular weight of 13,665 daltons. The translation start site was determined by N-terminal amino acid sequence analysis of a gcvH-lacZ encoded fusion protein. The E. coli H-protein shows extensive homology with the H-proteins from the pea (Pisum sativum) and the chicken liver GCV enzyme complexes. 85 of 128 amino acid residues are identical or chemically similar between the E. coli and the pea H-proteins, and 74 of 128 amino acid residues are identical or chemically similar between the E. coli and the chicken liver H-proteins. All three proteins have identical amino acid sequences from residues 61-65. This sequence contains the lysyl residue involved in lipoic acid attachment in the chicken liver H-protein.     

Purification and partial characterization of the glycine decarboxylase multienzyme complex from Eubacterium acidaminophilum.

The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum. By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an alpha 2 beta 2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively. In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity. All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction. The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate. The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2. Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2. Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity. Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.