The glycine cleavage system catalyzes the following reversible reaction: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 phosphatecontaining 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 -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.

Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of ␣-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 Å resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.Lipoic acid is a prosthetic group of acyltransferase (E2) subunit of the pyruvate, ␣-ketoglutarate, and branched-chain ␣-ketoacid dehydrogenase complexes and of H-protein of the glycine cleavage system (1-4). It attaches to a specific lysine residue on the proteins via an amide linkage between the ⑀-amino group of the lysine residue and the carboxyl group of lipoic acid. In the reaction sequence of the complexes, the lypoyllysine arm shuttles the reaction intermediates and reducing equivalents between the active sites of the components of the complexes.The attachment of lipoic acid to the proteins occurs by two-step reactions in which a lipoyl-AMP intermediate is formed from lipoic acid and ATP, and pyrophosphate is released in the initial activation reaction (Reaction 1).The lipoyl moiety of the intermediate is then transferred to apoproteins in the second transfer reaction, yielding the lipoylated protein and AMP (Reaction 2).Lipoyl-AMP ϩ apoprotein 3 lipoylated protein ϩ AMP (5, 6). LplA has a molecular mass of 37,795 Da, consisting of 337 amino acids excluding the initiating methionine residue, which is cleaved off during the biosynthesis (5). Strains with lplA null mutations have severe defects in the incorporation of exogenously supplied lipoic acid and lipoic acid analogues into apoproteins (5, 7). In E. coli, there is another enzyme, LipB, responsible for the covalent attachment of lipoic acid. LipB transfers lipoic acid/octanoic acid endogenously synthesized on the acyl carrier protein by the function of LipA to the lipoate-dependent enzymes (7-9). LipB consists of 213 amino acids, whose amino acid sequence shares only 12.7% identity with that of LplA (Fig. 1). On the other hand, the amino acid sequence of LplA shows 31 and 35% identity with those of human and bovine lipoyltransferase, the mammalian LplA homologues, respectively (Fig. 1). However, the mammalian lipoyltransferases have no ability to activate l...

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) -EJB 930930/1 P-protein, H-protein and T-protein of the glycine cleavage system have been purified from Escherichia coli. Their N-terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning. The nucleotide sequence of a fragment of DNA around the 62-min region of the E. coli chromosome, containing genes for the components of the glycinecleavage system has been determined. The sequence i?cludes three structural genes encoding Tprotein (363 amino acids, 40013 Da), H-protein (128 amino acids, 13679 Da) and P-protein (956 amino acids, 104240Da). These genes are named gcvT, gcvH and gcvP, respectively. They are organized in the above-mentioned order on the same strand of DNA with short intercistronic sequences. The presence of a potential promoter preceding gcvT and a typical rho-independent terminator sequence following gcvP indicated that the three genes constitute a single operon. Each component of the E. coli glycine-cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts. When the plasmid containing the gcv operon was transfected in E. coli cells, the gene products of gcvT gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon. However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5' terminal portion of gcvT failed to overexpress any of the three components.The glycine-cleavage system is a niultienzyme complex that catalyzes the reversible oxidation of glycine, yielding carbon dioxide, ammonia, methylenetetrahydrofolate and a reduced pyridine nucleotide [l -51. The enzyme system has been purified from a number of sources including animal liver [6], plants [7,8] and bacteria such as Pepfococcus glycinophilus [9], Arthrobacter globformis [lo] and Eubacterium acidaminophilum [ll]. The system is composed of four proteins named p-, H-, T-and L-protein In Escherichia coli the glycine-cleavage system is inducible by exogenous glycine [24] and encoded by the gcv structural genes situated at 62 min in the linkage map [25]. Although a gene for H-protein has been sequenced [26], the detailed properties of the glycine-cleavage system in E. coli have not been examined so far. Our purpose is to characterize the structural genes for the components of the glycine-cleavage system from E. coli and to acquire more information about the characteristic features of the deduced amino acid sequences and the binding sites for coenzymes and prosthetic groups. In this study, we report the molecular cloning of these genes, their nucleotide sequences and deduced amino acid sequences and evidence for their arrangement in an operon. Where possible, these proteins have been compared to their animal and plant counterparts.

In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg 2؉ ion, and the reaction product was lipoyl-GMP. LAE activated both (R)-and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)-and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.