Fig 1.
Enzyme reaction and amino acid sequence alignment of CgAspAT.
(A) Enzyme reaction of CgAspAT. (B) Amino acid sequence alignment of AspATs from subgroup Ic. The secondary structural elements are drawn based on the structure of CgAspAT. Residues involved in the binding of the PLP cofactor and the glutamate substrate are indicated by red- and green-colored triangles. The core domain and auxiliary domain are indicated as core and auxiliary, respectively. Cg, Cd, Mt and Dg are abbreviations of Corynebacterium glutamicum, Corynebacterium diphtheria, Mycobacterium tuberculosis and Deinococcus geothermalis, respectively. ‘Clustal Omega’ and ‘ESPript’ software were used for sequencing alignment.
Table 1.
Data collection and refinement statistics.
Fig 2.
(A) The monomeric structure of CgAspAT. The monomeric structure of CgAspAT is presented as a cartoon diagram. The core domain and the auxiliary domain are distinguished with orange and light-blue colors, respectively, and labeled. The bound PLP cofactor is shown as a magenta-colored sphere model. Secondary structure elements are labeled. (B) Dimeric structure of CgAspAT. The dimeric structure of CgAspAT is presented as a cartoon diagram. The bound PLP cofactor is shown as a magenta-colored sphere model. The bottom figure is rotated by 90 degree from the top.
Fig 3.
(A) Electron density map of the bound PLP. The 2Fo-Fc electron density map of the bound PLP in CgAspAT is shown with a gray-colored mesh, and contoured at 1.2 σ. (B) PLP binding mode of CgAspAT. The bound PLP cofactor is presented as a stick model with a magenta color. Residues involved in the binding of PLP are shown as stick models and labeled appropriately. The core domain and the auxiliary domain of one molecule are distinguished with orange and light-blue colors, respectively, and the other molecule is with a cyan color. Hydrogen bonds involved in the PLP binding are shown with red-colored dotted lines. (C) Site-directed mutagenesis of CgAspAT. Residues involved in binding of cofactor and substrate are replaced by alanine residues. The relative activities of recombinant mutant proteins were measured and compared with that of wild-type CgAspAT.
Fig 4.
Substrate binding mode of CgAspAT.
(A) Electron density map of the bound citrate in CgAspAT. The electron density maps Fo-Fc of the bound citrate in CgAspAT is shown with a gray-colored mesh, and contoured at 2.5 σ (B) Electrostatic potential surface model of the substrate binding site. The CgAspAT structure is presented as an electrostatic potential surface model. The bound PLP and citrate are shown as stick models with cyan and orange colors, respectively. The highly positively-charged residues constituting the substrate binding site are labeled. (C) Stereo-view of substrate binding mode of CgAspAT. The CgAspAT structure is superposed with the EcAspAT structure in complex with PLP-glutamate. Structures of CgAspAT and EcAspAT are shown as cartoon diagram with magenta and green colors, respectively. Residues involved in the glutamate binding are shown as a stick model and labeled appropriately. The PLP-glutamate bound in EcAspAT is shown as a stick model with a yellow color, and the PLP molecule and the citrate ion bound in CgAspAT are with colors of cyan and orange, respectively.
Fig 5.
Comparison of CgAspAT with subgroup Ia and Ib.
(A) Comparison of the auxiliary domain of CgAspAT with those of AspATs from E. coli (EcAspAT, subgroup Ia) and T. thermophilus (TtAspAT, subgrpup Ib). Structures of EcAspAT, TtAspAT and CgAspAT are superimposed. The core domains of these enzymes are shown with a gray color, and the auxiliary domains of EcAspAT, TtAspAT and CgAspAT are distinguished with green, orange and magenta colors, respectively. The bound PLP and glutamate in CgAspAT are shown as sphere model with magenta and cyan colors, respectively. RMSD values for EcAspAT-TtAspAT, EcAspAT-CgAspAT, and TtAspAT-CgAspAT are 2.73, 3.39, and 2.93, respectively. EcAspAT, TtAspAT and CgAspAT are representative AspATs for subgroup Ia, Ib and Ic, respectively. (B) Comparison of the cofactor binding mode of EcAspAT, TtAspAT and CgAspAT. Structures of EcAspAT, TtAspAT and CgAspAT are superimposed. Residues involved in the PLP cofactor binding are shown as a stick model with green, orange and magenta colors for EcAspAT, TtAspAT and CgAspAT, respectively. The bound PLP in CgAspAT is shown as a stick model with a cyan color. (C) Comparison of the substrate binding mode of EcAspAT, TtAspAT and CgAspAT. Structures of EcAspAT, TtAspAT and CgAspAT are superimposed. Residues involved in the substrate binding are shown as a stick model with green, orange and magenta colors for EcAspAT, TtAspAT and CgAspAT, respectively. The bound PLP-glutamate in EcAspAT is shown as a stick model with a yellow color. (D) Comparison of the auxiliary domain of AspATs from subgroup Ic. Structures of CgAspAT, CdAspAT, MtAspAT and DgAspAT are superimposed. The core domains of these enzymes are shown with a gray color, and the auxiliary domains of CgAspAT, CdAspAT, MtAspAT and DgAspAT are distinguished with cyan, orange, light blue and magenta colors, respectively. The bound PLP and glutamate in CgAspAT are shown as stick models with magenta and cyan colors, respectively. RMSD values for CgAspAT-CdAspAT, CgAspAT-MtAspAT, CgAspAT-DgAspAT, CdAspAT-MtAspAT, CdAspAT-DgAspAT, and MtAspAT-DgAspAT are 0.65, 0.12, 1.33, 0.66, 1.38, and 1.35, respectively. (E) Comparison of the cofactor binding mode of AspATs from subgroup Ic. Structures of CgAspAT, CdAspAT, MtAspAT and DgAspAT are superimposed. Residues involved in the PLP cofactor binding are shown as a stick model with cyan, orange, light blue and magenta colors for CgAspAT, CdAspAT, MtAspAT and DgAspAT, respectively. The bound PLP in CgAspAT is shown as a stick model with a cyan color. (F) Comparison of the substrate binding mode of AspATs from subgroup Ic. Structures of CgAspAT, CdAspAT, MtAspAT and DgAspAT are superimposed. Residues involved in the substrate binding are shown as a stick model with cyan, orange, light blue and magenta colors for CgAspAT, CdAspAT, MtAspAT and DgAspAT, respectively. The bound citrate in CgAspAT is shown as a stick model with an orange color.
Fig 6.
Phylogenetic relationships between AspAT enzymes.
The maximum likelihood tree was calculated using MEGA (Molecular Evolutionary Genetics Analysis) software on the basis of a Clustal Omega. Alignment of AspAT enzymes from three different subgroups. Ten, five, and four AspAT enzymes are selected for subgroup Ia, Ib, and Ic, respectively.