PMC

Overall structure of the Synechocystis P-protein homodimer in the reduced state with the cofactor PLP bound. Shown is a ribbon diagram with β-sheets in yellow and helices in purple in one subunit and green in the second subunit. PLP is shown as red spheres.

Electrostatic surface analysis of apoenzyme (A) and holoenzyme (B). Electrostatic surfaces are colored blue in the positive region and red in the negative region. The C terminus and the mobile loop are shown in a black ribbon representation. PLP is indicated as red spheres in B.

Conformational states of the C terminus in oxidized and reduced P-protein. Superposition of ribbon models of the oxidized, ligand-free P-protein (beige) and the reduced, PLP-bound P-protein (light blue) is shown. The C terminus (residues 962–978) of the oxidized and reduced P-protein is highlighted in red and blue, respectively. Sulfur atoms of cysteines 353 and 972 are colored green. PLP (bound in the reduced enzyme) is shown as spheres with carbon atoms colored yellow.

Stereo image showing a comparison of the homodimeric and heterotetrameric P-protein structures. The T. thermophilus heterodimer () in blue and red is superimposed on a monomer of the Synechocystis enzyme in white. The loop, residues 461–494, connecting the N-terminal and C-terminal parts of the Synechocystis monomer is colored green, and PLP is shown as yellow spheres. N-terminal and C-terminal residues of the α- and β-chains of the T. thermophilus enzyme are labeled in blue and red, respectively.

Specific activities of P-protein variants at different redox conditions relative to the wild-type protein. The columns show mean activities of recombinant P-protein ± S.D. (error bars) (n = 4) preincubated with 1 mm (black) or zero (gray) dithiothreitol in the glycine-bicarbonate exchange reaction. The numbers indicate percentage activities relative to the corresponding wild-type control. The number in parentheses shows the percentage relative to fully activated wild-type P-protein.

Binding of PLP and glycine in the active site of P-protein. A, residues in contact with PLP are shown as white sticks with hydrogen bonds indicated by red dashed lines. PLP (yellow) is covalently bound to Lys⁷²⁶. B, σA weighted difference density map (mF_o − DF_c) drawn at the level of 3 times the r.m.s. value of the map around PLP (yellow) and glycine (cyan) with hydrogen bonds indicated by red dashed lines. Water molecules are shown as red spheres.

Conformation of the mobile loop and the C terminus in the homodimeric P-protein. A, ribbon model of the apo-P-protein (beige) with the C terminus (residues 962–978) and the mobile loop (residues 335–353) highlighted in red and pink, respectively. The disulfide bond between cysteines 353 and 972 is colored green. B, holo-P-protein (light blue ribbon) with PLP and glycine bound. The C terminus and the mobile loop are highlighted in blue and cyan, respectively, and PLP and glycine are shown as spheres. C, superposition of the models in A and B showing the overlap of the positions of the closed C terminus and the closed mobile loop.

Schematic view of the reactions catalyzed by the GCS. A, the overall reaction catalyzed by the P-, T-, L-, and H-proteins of the GCS. First, the P-protein catalyzes the decarboxylation of glycine and the transfer of the aminomethylene moiety to the oxidized lipoamide arm of the H-protein. Next, the T-protein catalyzes the transfer of the methylene group to THF, thereby releasing NH₃. Finally, the L-protein reoxidizes the reduced lipoamide group of the H-protein. B, outline of the involvement of the PLP cofactor in the decarboxylation reaction catalyzed by the P-protein. PLP first forms an internal aldimine with a lysine residue in the active site (Lys⁷²⁶ in the Synechocystis enzyme). Next the C4′ atom of PLP is attacked by glycine to form the external aldimine, thereby releasing the lysine. Finally CO₂ is released from the external aldimine, leaving a quinoid intermediate ready to bind lipoamide.

Amino acid sequence alignment of P-proteins based on a structural alignment of the enzymes from Synechocystis sp. PCC 6803 and T. thermophilus (PDB code 1WYV). The sequences were obtained from the NCBI database. The sequence of Synechocystis P-protein was compared with homologous proteins from A. thaliana, Homo sapiens, and T. thermophilus. Residue numbering is according to the Synechocystis sequence. Because the N-terminal target peptides were omitted, the residue numbering of A. thaliana P-protein starts from position 40, and the H. sapiens numbering starts from position 57. To facilitate alignment, peptides α^N and β^C of T. thermophilus were fused to one polypeptide. Conserved residues are boxed, strictly conserved residues have a red background, residues well conserved within a group are indicated by red letters, and the remainder are in black letters. Symbols above blocks of sequences correspond to the secondary structure of the Synechocystis enzyme: α, α-helix; β, β-strand; η, 3₁₀-helix. Gray asterisks above the sequence indicate the presence of alternate conformations. The secondary structure assignment for the Synechocystis enzyme was made using DSSP (). The figure was prepared using ESPript ().