Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase

Abstract

The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ΔG(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.

Toward rational thermostabilization of Aspergillus oryzae cutinase: Insights into catalytic and structural stability

“…This is explained by that the mutations improved the stability of wt‐AoC at locations that are sufficiently distant from the active site such that CD spectra show that the overall structure of the protein is stabilized. Similar observations are reported for the LCC where the difference between the thermal unfolding temperature and the optimum temperature for activity is remarkably high, that is, 36°C which was again explained based on the lower active site stability as compared with the global structural stability …”

“…Most of the known cutinases are stable from 45°C to 50°C with the exception of a few produced by thermophilic microorganisms, which are stable in the range of 55°C–70°C, for example, H. insolens ( T m = 63°C), T. fusca ( T m = 70°C), T. alba ( T m = 60°C) and a mesophilic fungi A. oryzae ( T m = 62°C) . Recently a cutinase identified from leaf branch compost using a metagenomics approach was reported to be highly thermostable with the T m of 83°C, however the enzyme suffers lower kinetic stability at higher temperature ( t 1/2 of 40 minutes at 70°C) at the same time loses activity at temperatures higher than 50°C . Given the commercial importance of catalysis at or exceeding T g for polymers such as PBT and PET to allow efficient surface modification and mild recycling to monomers, this article is focused on thermostabilization of the cutinase from AoC.…”

Biodegradation of diethyl terephthalate and polyethylene terephthalate by a novel identified degraderDelftiasp. WL-3 and its proposed metabolic pathway

“…Undoubtedly, both fungi and bacteria can transform PET into terephthalic acid (TPA) via the same degradation pathway under the catalysis of esterases and cutinases (Sulaiman et al . ; Wei et al . ).…”

Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris cutinase biochemical characteristics

“…These enzymes are structurally distinct from FsC but do have structural homology to lipases from the genus Streptomyces . Recently, a cutinase isolated from leaf and branch compost with structural similarity to T. alba cutinase was characterized and found to be highly thermostable ( T m = 86 °C) (Sulaiman et al 2012, 2014). …”