Structural basis for Ca2+-dependent catalysis of a cutinase-like enzyme and its engineering: application to enzymatic PET depolymerization

Abstract

A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, can depolymerize polyethylene terephthalate (PET). As high activity at approximately 70°C is required for PET depolymerization, structure-based protein engineering of Cut190 was carried out. Crystal structure information of the Cut190 mutants was used for protein engineering and for evaluating the molecular basis of activity and thermal stability. A variety of biophysical methods were employed to unveil the mechanisms underlying the unique features of Cut190, which included the regulation of its activity and thermal stability by Ca²⁺. Ca²⁺ association and dissociation can change the enzyme conformation to regulate catalytic activity. Weak metal-ion binding would be required for the naïve conformational change of Cut190, while maintaining its fluctuation, to "switch" the enzyme on and off. The activity of Cut190 is regulated by the weak Ca²⁺ binding to the specific site, Site 1, while thermal stability is mainly regulated by binding to another Site 2, where a disulfide bond could be introduced to increase the stability. Recent results on the structure-activity relationship of engineered Cut190 are reviewed, including the application for PET depolymerization by enzymes.

Keywords: crystal structure; metal-ion binding; polyethylene terephthalate; thermal stability.

Figure 1

The amino acid sequence of Cut190 and Cut190 mutants designated. The regions of helices and β-sheets are underlined in wavy and straight lines, respectively.

Figure 2

Crystal structures of Cut190 mutants. The structures of closed form (pink, PDB ID: 4WFI), open forms (without substrate; green, PDB ID: 5ZRQ, with substrate; purple, PDB ID: 5ZRS), engaged form (cyan; PDB ID: 5ZRR), and ejecting form (yellow, PDB ID: 7CEH) are superimposed. The figures were generated using the program PyMOL. (A) Overall structures. Spheres indicate bound metal ions. The side chains of Phe77, Phe81, Ser176, Asp222, His254 (PDB ID: 4WFI) and Phe106, Met177, Pro226, Arg228 (PDB ID: 5ZRQ) are also indicated as stick models. (B) Close-up view at β3-α2 and β4-α3 loops. The side chains of Phe106 and Thr107 are indicated as stick models and the substrate, monoethyl succinate, is indicated as a sphere model. (C) Close-up view at β1-β2 loop. The side chains of Phe77 and Phe81 are indicated as stick models.

Figure 3

ITC profiles of metal-ion binding to Cut190*S176A at 25°C. A solution of ZnCl₂ (A), MnCl₂ (B), MgCl₂ (C), and CaCl₂ (D) was injected into Cut190*S176A on iTC200 calorimeter. The K_a values are also indicated.

Figure 4

Crystal structures of Cut190*SS_S176A (green, PDB ID: 7CTS), LCC mutant Y127G/S165A/D238C/F243I/S283C (cyan, PDB ID: 6THT), and Cut190** (purple, PDB ID: 7CEF). A sphere indicates Ca²⁺ bound to Cut190*SS_S176A. The side chains of His123, Ala138, His202 and the disulfide bond of Cut190*SS_S176A are indicated as stick models. The figure was generated using the program PyMOL.