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

Abstract

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 Arg(292) 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.

FIGURE 1.

Outline of the reversible reaction of the GCS. P, T, L, and H are the protein components of GCS. Hox, Hint, and Hred represent H-proteins bearing covalently attached lipoate (oxidized form), aminomethyllipoate, and dihydrolipoate, respectively.

FIGURE 2.

Interface structure of the heterodimer of the ecT·ecHred complex. A, schematic representation of the ecT·ecHred heterodimer (MolAE). Domains 1, 2, and 3 of ecT are colored in light blue, cyan, and magenta, respectively, and ecHred is shown in green with the dihydrolipoyllysine arm shown in CPK model colored in green (carbon), blue (nitrogen), red (oxygen), and orange (sulfur). The bound 5-CH₃-THF is also shown in a CPK model colored in yellow (carbon) and the same colors for other atoms as for dihydrolipoyllysine. B, residues contributing to the ecT and ecHred interface. Hydrogen bonding and hydrophobic interactions are summarized. C, close-up view of the interface of molAE in stereo. Key residues from ecT (molA) and ecHred (molE) are shown as sticks with carbon atoms colored in light blue and pink for ecT and green for ecHred. The dihydrolipoyllysine arm is also in stick colored as in A. Hydrogen bonds are depicted as broken lines. D, glycine cleavage (GC) and synthesis (GS) activities of wild-type (WT) and R292A mutant ecT in the overall reaction assays. The inset depicts the trace activities by a large amount of the mutant. E, evaluation of the heterodimer-forming ability of ecT mutant. ecT or ecTR292A was mixed with 1.5–2-fold molar excess of ecHred and subjected to gel filtration. ecT was eluted at the same position of ecTR292A.

FIGURE 3.

Active site of ecT in complex with ecHred and 5-CH₃-THF. A, molecular surface of the active site pocket of ecT with the dihydrolipoyllysine arm inserted. Red and blue surfaces indicate negatively and positively charged areas, respectively. ecH is shown schematically, colored in green, and the dihydrolipoyllysine and 5-CH₃-THF are in stick colored as in Fig. 1A. The residues of ecT interacting with the dihydrolipoyllysine arm were shown in stick with labels. B, distances between the tip of dihydrolipoate and the atoms of folate cofactors. 5,10-CH₂-THF (magenta) was modeled based on the structure of 5-CH₃-THF (yellow). Distances (Å) are shown with broken lines. C, close-up view of the active site in stereo. Key residues and four water molecules are depicted in stick (cyan and light blue) and sphere (red), respectively, with labels. The dihydrolipoyllysine and 5-CH₃-THF are as in A, and hydrogen bonds are drawn as broken lines. D, putative ammonium-binding site in the reverse reaction. Omit F_o − F_c electron density for the water molecules at the active site of molAE is shown in green, contoured at 2.0σ. Well ordered W868 may occupy the ammonium-binding site. E, ecHint cleavage activity of wild-type and mutant ecTs in the absence of THF. The reaction was carried out as described under “Experimental Procedures.” Mock means nonenzymatical degradation of ecHint. Error bars show variability among three independent measurements.

FIGURE 4.

Structural comparison of huT with ecT. A, NKH-related mutation sites mapped on the overall topology of superimposed huT and ecT. The structure of huT (PDB code 1WSV, red) is overlaid on that of ecT (blue) of the ecT·ecHred complex and represented in ribbon. Mutation residues of huT and the corresponding residues of ecT are shown in CPK and stick, respectively, with numbers for huT. ecHred is shown in ribbon colored in green with dihydrolipoyllysine in stick. 5-CH₃-THF is shown in stick colored in yellow. B, alignment of key residues of huT and ecT. C, close-up view of the hydrogen bond networks assembling four highly conserved regions in T-protein. Key residues of huT (light blue) and ecT (pink) contributing to the assembly are represented in stick with numbers for huT. Mutant residues are depicted in ball-and-stick representation with atoms colored in red (carbon and oxygen) and blue (nitrogen) with red labels. Four highly conserved regions in the main chain of huT represented schematically (residues 51–56, 196–204, 229–248, and 229–328) are colored in red. The structure of ecHred of the complex and its residues are shown in schematically and stick, respectively, colored in green. Hydrogen bonds are drawn in broken lines.

FIGURE 5.

Model of the reaction mechanism for T-protein catalysis. The mechanism proposed for the forward (A) and reverse (B) reaction is represented schematically.