Enhanced enzyme kinetic stability by increasing rigidity within the active site

Abstract

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

Keywords: Active Site; Crystal Structure; Lipase; Local Rigidity; Mutagenesis; Protein Design; Protein Stability.

FIGURE 1.

CalB sites chosen for iterative saturation mutagenesis. The diagram shows a structural model based on the x-ray crystallographic structure of CalB (Protein Data Bank code 1TCA). Mutation sites around the catalytic Ser¹⁰⁵ residue are shown in cyan: Leu²⁷⁸, Ile²⁸⁵, Leu²⁷⁷, Ala²⁸¹, Phe⁷¹, and Asp²²³. Mutation sites on the protein surface are shown in green: Arg²⁴⁹, Arg³⁰⁹, Arg²⁴², Glu²⁶⁹, Pro²¹⁸, Leu²¹⁹, and Lys¹³. The B factor and distances between the side chains of selected residues and Ser¹⁰⁵ residue were measured. The Ser¹⁰⁵ residue is shown in stick form, and the area within 10 Å of this residue is outlined with a red circle.

FIGURE 2.

Thermally and urea-induced inactivation/unfolding profiles of wild-type and mutant CalB. A, thermal inactivation profiles of CalB mutants. Enzymes in 50 mm sodium phosphate buffer, pH 7.5 were incubated at various temperatures for 15 min and assayed for residual activity at 37 °C. The activity measured at 37 °C was considered to be 100%. B, results from differential scanning calorimetric analysis of the CalB mutants. The scans were analyzed after subtraction of an instrument-derived baseline recorded with buffer in both cells as calculated using the Origin software package. C, the fluorescence spectra were recorded at wavelengths between 300 and 400 nm with an excitation wavelength at 282 nm under a scanning speed of 1200 nm/min. D, urea-induced inactivation profiles of wild-type and mutant CalB. Enzymes were incubated with different concentrations of urea for 24 h and then assayed for residual activity at 37 °C. Activity measured in the absence of urea was considered to be 100%. ■, wild type; ●, D223G; ▴, L278M; ▾, D223G/L278M. The fitted curves for protein unfolding are shown as black lines. Error bars represent S.D.

FIGURE 3.

Temperature dependence of wild-type and mutant CalB. Enzymes were incubated at various temperatures for 1 min prior to activity measurement at the same temperature after the addition of substrate. The temperature-k_cat profile according to the Arrhenius plot is also shown (inset). ■, wild type; ●, D223G; ▴, L278M; ▾, D223G/L278M. Error bars represent S.D.

FIGURE 4.

Three-dimensional structure superimposition and hydrogen bond analysis in the CalB mutants. A, structure alignment of wild-type (white) and mutant CalB. The D223G, L278M, and D223G/L278M mutants are depicted in red, green, and magenta, respectively. Crucial amino acids are shown as lines, whereas Ser¹⁰⁵ residue and the D223G and L278M mutants are shown in stick form. B, crucial amino acids in wild-type CalB. C, crucial amino acids in L278M. D, crucial amino acids in D223G/L278M. E, interactions between amino acid 278 and other residues in wild-type CalB and the D223G/L278M mutant. Crucial amino acids are shown in ball and line, whereas L278/M278 is shown in stick form. Yellow dashed lines indicate the extended network of hydrogen bonds.

FIGURE 5.

Relative B factor profiles for wild-type (black) and D223G/L278M (magenta) CalB. With the highest residue B factor set at 100%, the relative residue B factors for the entire protein were calculated. Structures of the 257–281 segment for wild-type CalB and D223G/L278M mutant are shown. The crucial amino acids are shown with lines, and the extra network of hydrogen bonds is shown as yellow dashed lines.

FIGURE 6.

RMSD values during a 30-ns MD simulation for wild-type and mutant CalB. RMSD profiles of wild-type (A), D233G (B), L278M (C), and D233G/L278M (D) CalB at 300 and 330 K. RMSD values at 300 and 330 K are shown in black and red, respectively.

FIGURE 7.

RMSF values during a 30-ns molecular dynamic simulation for wild-type and D223G/L278M CalB at 300 and 330 K. A, RMSF profile of wild-type CalB at 300 and 330 K. B, RMSF profile of D223G/L278M at 300 and 330 K. RMSF values at 300 and 330 K are shown in black and red, respectively.