Table 1.
Data collection and refinement statistics of SrLDCA225C/T302C.
Fig 1.
Rationale of introduction of an artificial disulfide bond in SrLDC.
(A) Open/closed conformational changes at the PLP binding site of SrLDC. The open and the closed conformations of SrLDC are superimposed and shown as a cartoon diagram with green and magenta colors, respectively. The right side figure is an amplification of the dotted-box of the left figure. The AS-loop, PS-loop and R-loop of the open and the closed conformations of SrLDC are labeled. The bound PLP and cadaverine are shown as stick models and labeled. The target residues for the introduction of a disulfide bond are indicated with dotted-circle. (B) Schematic of the introduced disulfide bonds in SrLDC. The sheet domain and barrel domain are presented with green and purple colors, respectively. The residues replaced by cysteines to introduce a disulfide bond are presented with black line and labeled. (C) (D) A rationale of introduction of a disulfide bond. The open and the closed conformations of SrLDC are superimposed and shown as a cartoon diagram. The R-loop of the open and closed conformation of SrLDC are presented with cartoon diagrams with the same color scheme with (A). The residues selected to generate the SrLDCA225C/T302C mutant (C) and the SrLDCK2C/G227C mutant (D) are shown as stick models and labeled.
Fig 2.
Enhanced PLP affinity in the SrLDCA225C/T302C mutant.
(A) Purified recombinant SrLDC proteins. The purified SrLDCWT, SrLDCK2C/G227C, and SrLDCA225C/T302C proteins with concentration of 1 mg/ml are shown. (B) UV/vis spectra of recombinant SrLDC proteins. The UV/Vis absorption spectra of recombinant SrLDC proteins are shown and labeled appropriately. The band peaks of 327 nm and 418 nm observed in the SrLDCA225C/T302C mutant are indicated. (C) Isothermal titration calorimetry of SrLDCWT and SrLDCA225C/T302C mutant. Kd values of SrLDCWT and the SrLDCA225C/T302C mutant are displayed.
Fig 3.
Comparison of LDC activity between SrLDC proteins
(A) Comparison of LDC activities of the SrLDC proteins. LDC activities of SrLDCWT, SrLDCK2C/G227C, and SrLDCA225C/T302C are measured at various concentrations of PLP. (B) Comparison of cadaverine conversion of the SrLDC proteins. Cadaverine conversion of SrLDCWT, SrLDCK2C/G227C, and SrLDCA225C/T302C are measured at various concentrations of PLP. (C) Comparison of time-course cadaverine conversion between SrLDCWT and SrLDCA225C/T302C. Time-course cadaverine conversion of SrLDCWT and the SrLDCA225C/T302C mutant are measured under PLP concentrations of 0.02 and 0.2 mM. (D) Comparison of resistance to pH between SrLDCWT and SrLDCA225C/T302C. The LDC activity of SrLDCWT is measured at pH from 5 to 10 under 0.2 mM PLP. The LDC activities of the SrLDCA225C/T302C mutant are also measured at the same pH range under 0.01 and 0.2 mM PLP.
Fig 4.
Comparison of resistance to temperature between SrLDCWT and SrLDCA225C/T302C.
(A) Thermal stability measurement of SrLDCWT and SrLDCA225C/T302C. The Tm values of SrLDCWT and the SrLDCA225C/T302C mutant are displayed. (B) (C) Comparison of thermal stability between SrLDCWT and SrLDCA225C/T302C. The LDC activities of SrLDCWT and the SrLDCA225C/T302C mutant are measured using proteins pre-incubated at 37°C (B) and 60°C (C).
Fig 5.
Crystal structure of the SrLDCA225C/T302C mutant.
(A) Dimeric structure of the SrLDCA225C/T302C mutant. The AS-loop, PS-loop and the R-loop of the SrLDCA225C/T302C mutant are colored green, salmon, and light-blue, respectively. The disulfide bond formed in both monomers are indicated are shown as stick model and labeled. (B) Electron density map of the engineered disulfide bond region. The Fo-Fc map of the engineered disulfide bond region is contoured at 3.0 σ. The R-loop region is presented with a light-blue color and the mutated residues are shown as stick models with labels. (C) Superimposition of the SrLDCA225C/T302C structure with the closed form of SrLDC. The AS-loop, the PS-loop, and the R-loop of the SrLDCA225C/T302C mutant are distinguished with colors of green, salmon, and light-blue, respectively, and those of the closed form of SrLDC are with colors of orange, cyan, and magenta, respectively. (D) B-factor presentation of the SrLDCA225C/T302C mutant and SrLDC in apo and complex with PLP/cadaverine. The AS-loop, the PS-loop, and the R-loop are labeled.