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.     

Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase

Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes dimethylglycine to a labile iminium intermediate. The C-terminal region binds tetrahydrofolate, comprises three domains arranged in a ring-like structure and is related to the T-protein of the glycine cleavage system. The complex with folinic acid indicates that this enzyme selectively activates the N10 amino group for initial attack on the substrate. Dead-end reactions with oxidized folate are avoided by the strict stereochemical constraints imposed by the folate-binding funnel. The active sites in DMGO are approximately 40 A apart, connected by a large irregular internal cavity. The tetrahydrofolate-binding funnel serves as a transient entry-exit port, and access to the internal cavity is controlled kinetically by tetrahydrofolate binding. The internal cavity enables sequestration of the reactive iminium intermediate prior to reaction with tetrahydrofolate and avoids formation of toxic formaldehyde. This mode of channelling in DMGO is distinct from other channelling mechanisms.     

Heterotetrameric sarcosine oxidase: structure of a diflavin metalloenzyme at 1.85 A resolution

The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.     

Genomic structures and sequences of two closely linked genes (AMT, TCTA) on dog chromosome 20q15.1-->q15.2

Analysis of genomic sequence from canine chromosome 20q15.1-->q15.2 revealed the presence of two closely linked genes. The two genes represent the corresponding canine orthologs of human aminomethyltransferase (AMT) and the human T-cell leukemia translocation associated (TCTA) gene. Aminomethyltransferase or glycine cleavage system T-protein is an important enzyme in glycine metabolism. The reported canine AMT gene spans 5 kb and consists of nine exons. It encodes a protein of 403 amino acids with 88% identity to human aminomethyltransferase. Human TCTA is located on 3p21 near the breakpoint of a t(1;3) translocation observed in some cancer cell lines. The 4-kb canine TCTA gene consists of three exons and probably represents a pseudogene. It is located adjacent to AMT and very close to DAG1 and BSN.     

Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia

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-CH3-H4folate) 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-CH3-H4folate 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 alpha-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-CH3-H4folate, 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.     

Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing effects on growth and on activity of the iron-sulfur enzyme MiaB

The folate-dependent protein YgfZ of Escherichia coli participates in the synthesis and repair of iron-sulfur (Fe-S) clusters; it belongs to a family of enzymes that use folate to capture formaldehyde units. Ablation of ygfZ is known to reduce growth, to increase sensitivity to oxidative stress, and to lower the activities of MiaB and other Fe-S enzymes. It has been reported that the growth phenotype can be suppressed by disrupting the tRNA modification gene mnmE. We first confirmed the latter observation using deletions in a simpler, more defined genetic background. We then showed that deleting mnmE substantially restores MiaB activity in ygfZ deletant cells and that overexpressing MnmE with its partner MnmG exacerbates the growth and MiaB activity phenotypes of the ygfZ deletant. MnmE, with MnmG, normally mediates a folate-dependent transfer of a formaldehyde unit to tRNA, and the MnmEG-mediated effects on the phenotypes of the ΔygfZ mutant apparently require folate, as evidenced by the effect of eliminating all folates by deleting folE. The expression of YgfZ was unaffected by deleting mnmE or overexpressing MnmEG or by folate status. Since formaldehyde transfer is a potential link between MnmEG and YgfZ, we inactivated formaldehyde detoxification by deleting frmA. This deletion had little effect on growth or MiaB activity in the ΔygfZ strain in the presence of formaldehyde, making it unlikely that formaldehyde alone connects the actions of MnmEG and YgfZ. A more plausible explanation is that MnmEG erroneously transfers a folate-bound formaldehyde unit to MiaB and that YgfZ reverses this.     

Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism

Proteins of the YgfZ family occur in all domains of life and are characterized by the conserved dodecapeptide motif KGC[Y/F]-x-GQE-x(3) -[R/K]. YgfZ proteins are known to participate in assembly or repair of iron/sulphur clusters, and to require folate for biological activity, but their mechanism of action is unknown. To assess the importance of individual residues in the conserved motif, Escherichia coli Ygf?Z was expressed from a plasmid in a ΔygfZ strain and subjected to alanine-scanning mutagenesis. The impacts on YgfZ functionality were evaluated by assays of growth and of the in vivo activity of the iron/sulphur enzyme MiaB, which modifies tRNA. By these criteria, the motif's tyrosine residue (Y229) had a detectable influence but only the cysteine residue (C228) was critical, for only the C228A mutant failed to complement the growth and MiaB activity phenotypes of the ΔygfZ strain. Immunoblots confirmed that the latter result was not simply because of a low level of the C228A mutant protein. Collectively, these data demonstrate a pivotal role for the Ygf?Z motif's cysteine residue and a subsidiary one for the adjacent tyrosine, and help formulate a hypothesis about the folate requirement of Ygf?Z proteins.     

Probing the H-protein-induced conformational change and the function of the N-terminal region of Escherichia coli T-protein of the glycine cleavage system by limited proteolysis

T-protein, a component of the glycine cleavage system, catalyzes a tetrahydrofolate-dependent reaction. Previously, we reported a conformational change of Escherichia coli T-protein upon interacting with E. coli H-protein (EH), showing an important role for the N-terminal region of the T-protein in the interaction. To further investigate the T-protein catalysis, the wild type (ET) and mutants were subjected to limited proteolysis. ET was favorably cleaved at Lys(81), Lys(154), Lys(288), and Lys(360) by lysylendopeptidase and the cleavages at Lys(81) and Lys(288) were strongly prevented by EH. Although ET was highly resistant to trypsinolysis, the mutant with an N-terminal 7-residue deletion (ETDelta7) was quite susceptible and instantly cleaved at Arg(16) accompanied by the rapid degradation of the resulting C-terminal fragment, indicating that the cleavage at Arg(16) is the trigger for the C-terminal fragmentation. EH showed no protection from the N-terminal cleavage, although substantial protection from the C-terminal fragmentation was observed. The replacement of Leu(6) of ET with alanine resulted in a similar sensitivity to trypsin as ETDelta7. These results suggest that the N-terminal region of ET functions as a molecular "hasp" to hold ET in the compact form required for the proper association with EH. Leu(6) seems to play a central role in the hasp function. Interestingly, Lys(360) of ET was susceptible to proteolysis even after the stabilization of the entire molecule of ET by EH, indicating its location at the surface of the ET-EH complex. Together with the buried position of Lys(81) in the complex and previous results on folate binding sites, these results suggest the formation of a folate-binding cavity via the interaction of ET with EH. The polyglutamyl tail of the folate substrate may be inserted into the bosom of the cavity leaving the pteridine ring near the entrance of the cavity in the context of the catalytic reaction.     

Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system.

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) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km 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 alpha-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 alpha-carboxyl group of the second glutamate residue.     

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.     

Enzymatic properties of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are flavoproteins which catalyze the oxidative demethylation of dimethylglycine to sarcosine and sarcosine to glycine, respectively. During these reactions tightly bound tetrahydropteroylpentaglutamate (H4PteGlu5) is converted to 5,10-methylene tetrahydropteroylpentaglutamate (5,10-CH2-H4PteGlu5), although in the absence of H4PteGlu5, formaldehyde is produced. Single turnover studies using substrate levels of the enzyme (2.3 microM) showed pseudo-first-order kinetics, with apparent first-order rate constants of 0.084 and 0.14 s-1 at 23 and 48.3 microM dimethylglycine, respectively, for dimethylglycine dehydrogenase and 0.065 s-1 at 47.3 microM sarcosine for sarcosine dehydrogenase. The rates were identical in the absence or presence of bound tetrahydropteroylglutamate (H4PteGlu). Titration of the enzymes with substrate under anaerobic conditions did not disclose the presence of an intermediate semiquinone. The effect of dimethylglycine concentration upon the rate of the dimethylglycine dehydrogenase reaction under aerobic conditions showed nonsaturable kinetics suggesting a second low-affinity site for the substrate which increases the enzymatic rate. The Km for the high-affinity active site was 0.05 mM while direct binding for the low-affinity site could not be measured. Sarcosine and dimethylthetin are poor substrates for dimethylglycine dehydrogenase and methoxyacetic acid is a competitive inhibitor at low substrate concentrations. At high dimethylglycine concentrations, increasing the concentration of methoxyacetic acid produces an initial activation and then inhibition of dimethylglycine dehydrogenase activity. When these compounds were added in varying concentrations to the enzyme in the presence of dimethylglycine, their effects upon the rate of the reaction were consistent with the presence of a second low-affinity binding site on the enzyme which enhances the reaction rate. When sarcosine is used as the substrate for sarcosine dehydrogenase the kinetics are Michaelis-Menten with a Km of 0.5 mM for sarcosine. Also, methoxyacetic acid is a competitive inhibitor of sarcosine dehydrogenase with a Ki of 0.26 mM. In the absence of folate, substrate and product determinations indicated that 1 mol of formaldehyde and of sarcosine or glycine were produced for each mole of dimethylglycine or sarcosine consumed with the concomitant reduction of 1 mol of bound FAD.     

Crystal Structure of Aminomethyltransferase in Complex with Dihydrolipoyl-H-Protein of the Glycine Cleavage System: IMPLICATIONS FOR RECOGNITION OF LIPOYL PROTEIN SUBSTRATE,DISEASE-RELATED MUTATIONS,AND REACTION MECHANISM*

Aminomethyltransferase, a component of the glycine cleavage system termed T-protein, reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate. Several mutations in the human T-protein gene are known to cause nonketotic hyperglycinemia. Here, we report the crystal structure of Escherichia coli T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction. The structure of the complex shows a highly interacting intermolecular interface limited to a small area and the protein-bound dihydrolipoyllysine arm inserted into the active site cavity of the T-protein. Invariant Arg²⁹² of the T-protein is essential for complex assembly. The structure also provides novel insights in understanding the disease-causing mutations, in addition to the disease-related impairment in the cofactor-enzyme interactions reported previously. Furthermore, structural and mutational analyses suggest that the reversible transfer of the methylene group between the lipoate and tetrahydrofolate should proceed through the electron relay-assisted iminium intermediate formation.     

Crystal structure at 2.4 A resolution of E. coli serine hydroxymethyltransferase in complex with glycine substrate and 5-formyl tetrahydrofolate

Serine hydroxymethyltransferase (EC 2.1.2.1), a member of the alpha-class of pyridoxal phosphate enzymes, catalyzes the reversible interconversion of serine and glycine, changing the chemical bonding at the C(alpha)-C(beta) bond of the serine side-chain mediated by the pyridoxal phosphate cofactor. Scission of the C(alpha)-C(beta) bond of serine substrate produces a glycine product and most likely formaldehyde, which reacts without dissociation with tetrahydropteroylglutamate cofactor. Crystal structures of the human and rabbit cytosolic serine hydroxymethyltransferases (SHMT) confirmed their close similarity in tertiary and dimeric subunit structure to each other and to aspartate aminotransferase, the archetypal alpha-class pyridoxal 5'-phosphate enzyme. We describe here the structure at 2.4 A resolution of Escherichia coli serine hydroxymethyltransferase in ternary complex with glycine and 5-formyl tetrahydropteroylglutamate, refined to an R-factor value of 17.4 % and R(free) value of 19.6 %. This structure reveals the interactions of both cofactors and glycine substrate with the enzyme. Comparison with the E. coli aspartate aminotransferase structure shows the distinctions in sequence and structure which define the folate cofactor binding site in serine hydroxymethyltransferase and the differences in orientation of the amino terminal arm, the evolution of which was necessary for elaboration of the folate binding site. Comparison with the unliganded rabbit cytosolic serine hydroxymethyltransferase structure identifies changes in the conformation of the enzyme, similar to those observed in aspartate aminotransferase, that probably accompany the binding of substrate. The tetrameric quaternary structure of liganded E. coli serine hydroxymethyltransferase also differs in symmetry and relative disposition of the functional tight dimers from that of the unliganded eukaryotic enzymes. SHMT tetramers have surface charge distributions which suggest distinctions in folate binding between eukaryotic and E. coli enzymes. The structure of the E. coli ternary complex provides the basis for a thorough investigation of its mechanism through characterization and structure determination of site mutants.     

Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp.: implications for glycine betaine catabolism.

The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1-IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identified in the soxBDAG operon corresponding to the four subunits of heterotetrameric sarcosine oxidase by comparison with the N-terminal amino-acid sequences and the subunit relative molecular masses of the purified enzyme. Alignment of the deduced sarcosine oxidase amino-acid sequence with amino-acid sequences of functionally related proteins indicated that the arthrobacterial enzyme is highly homologous to sarcosine oxidase from Corynebacterium P-1. Deletion and expression analysis, and alignment of the deduced amino-acid sequence of the dmg gene, showed that dmg encodes a novel dimethylglycine oxidase, which is related to eukaryotic dimethylglycine dehydrogenase, and contains nucleotide-binding, flavinylation and folate-binding motifs. The recombinant dimethylglycine oxidase was purified to homogeneity and characterized. The DNA located upstream and downstream of both the soxBDAG and dmg genes is predicted to encode enzymes involved in the tetrahydrofolate-dependent assimilation of methyl groups. Based on the sequence analysis reported herein, pathways are proposed for glycine betaine catabolism in Arthrobacter species, which involve the identified folate-dependent enzymes.     

An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

We report a synthetic biology approach to demonstrate substrate channeling in an unusual bifunctional flavoprotein dimethylglycine oxidase. The catabolism of dimethylglycine through methyl group oxidation can potentially liberate toxic formaldehyde, a problem common to many amine oxidases and dehydrogenases. Using a novel synthetic in vivo reporter system for cellular formaldehyde, we found that the oxidation of dimethylglycine is coupled to the synthesis of 5,10-methylenetetrahydrofolate through an unusual substrate channeling mechanism. We also showed that uncoupling of the active sites could be achieved by mutagenesis or deletion of the 5,10-methylenetetrahydrofolate synthase site and that this leads to accumulation of intracellular formaldehyde. Channeling occurs by nonbiased diffusion of the labile intermediate through a large solvent cavity connecting both active sites. This central “reaction chamber” is created by a modular protein architecture that appears primitive when compared with the sophisticated design of other paradigm substrate-channeling enzymes. The evolutionary origins of the latter were likely similar to dimethylglycine oxidase. This work demonstrates the utility of synthetic biology approaches to the study of enzyme mechanisms in vivo and points to novel channeling mechanisms that protect the cell milieu from potentially toxic reaction products.     

Protein expression in response to folate stress in Escherichia coli.

Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.     

Folate in demethylation: The crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate

Dimethylglycine dehydrogenase (DMGDH) is a mammalian mitochondrial enzyme which plays an important role in the utilization of methyl groups derived from choline. DMGDH is a flavin containing enzyme which catalyzes the oxidative demethylation of dimethylglycine in vitro with the formation of sarcosine (N-methylglycine), hydrogen peroxide and formaldehyde. DMGDH binds tetrahydrofolate (THF) in vivo, which serves as an acceptor of formaldehyde and in the cell the product of the reaction is 5,10-methylenetetrahydrofolate instead of formaldehyde. To gain insight into the mechanism of the reaction we solved the crystal structures of the recombinant mature and precursor forms of rat DMGDH and DMGDH–THF complexes. Both forms of DMGDH reveal similar kinetic parameters and have the same tertiary structure fold with two domains formed by N- and C-terminal halves of the protein. The active center is located in the N-terminal domain while the THF binding site is located in the C-terminal domain about 40 Å from the isoalloxazine ring of FAD. The folate binding site is connected with the enzyme active center via an intramolecular channel. This suggests the possible transfer of the intermediate imine of dimethylglycine from the active center to the bound THF where they could react producing a 5,10-methylenetetrahydrofolate. Based on the homology of the rat and human DMGDH the structural basis for the mechanism of inactivation of the human DMGDH by naturally occurring His109Arg mutation is proposed.     

Glycine metabolism by rat liver mitochondria. Reconstruction of the reversible glycine cleavage system with partially purified protein components

An enzyme system which catalyzes the degradation of glycine to one carbon unit, ammonia, and carbon dioxide and the synthesis of glycine from these three substances has been isolated from rat liver mitochondria. The reversible glycine cleavage system is composed of four protein components named as P-, H-, L-, and T-protein, respectively. A procedure is described for the purification of P-protein which catalyzes the decarboxylation of glycine or its reverse reaction in the presence of H-protein, and for T-protein which participates in the formation of one carbon unit and ammonia or the reverse reaction. The procedure described leads to the isolation of a nearly homogeneous form of T-protein but P-protein still is heterogeneous. The molecular weight of T-protein, estimated by molecular sieve chromatography, is 33,000. Properties of the synthesis and cleavage reactions and the exchange of carboxyl group of glycine with bicarbonate are also presented.     

Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme.

BACKGROUND: Monomeric sarcosine oxidases (MSOXs) are among the simplest members of a recently recognized family of eukaryotic and prokaryotic enzymes that catalyze similar oxidative reactions with various secondary or tertiary amino acids and contain covalently bound flavins. Other members of this family include heterotetrameric sarcosine oxidase, N-methyltryptophan oxidase and pipecolate oxidase. Mammalian sarcosine dehydrogenase and dimethylglycine dehydrogenase may be more distantly related family members. RESULTS: The X-ray crystal structure of MSOX from Bacillus sp. B-0618, expressed in Escherichia coli, has been solved at 2.0 A resolution by multiwavelength anomalous dispersion (MAD) from crystals of the selenomethionine-substituted enzyme. Fourteen selenium sites, belonging to two MSOX molecules in the asymmetric unit, were used for MAD phasing and to define the local twofold symmetry axis for electron-density averaging. The structures of the native enzyme and of two enzyme-inhibitor complexes were also determined. CONCLUSIONS: MSOX is a two-domain protein with an overall topology most similar to that of D-amino acid oxidase, with which it shares 14% sequence identity. The flavin ring is located in a very basic environment, making contact with sidechains of arginine, lysine, histidine and the N-terminal end of a helix dipole. The flavin is covalently attached through an 8alpha-S-cysteinyl linkage to Cys315 of the catalytic domain. Covalent attachment is probably self-catalyzed through interactions with the positive sidechains and the helix dipole. Substrate binding is probably stabilized by hydrogen bonds between the substrate carboxylate and two basic sidechains, Arg52 and Lys348, located above the re face of the flavin ring.