Sulaiman, S., You, D.J., Eiko, K., Koga, Y., Kanaya, S.
Ligand
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Reference
Title: Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase Sulaiman S, You DJ, Kanaya E, Koga Y, Kanaya S Ref: Biochemistry, 53:1858, 2014 : PubMed
The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 A 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 degrees C were 86.2 degrees C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, DeltaG(H2O), was 41.8 kJ mol(-1) at 30 degrees C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 x 10(-6) s(-1) at 50 degrees 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 degrees C) was considerably lower than the T1/2 value. It increased by 10 degrees C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 degrees 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.
        
Title: Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach Sulaiman S, Yamato S, Kanaya E, Kim JJ, Koga Y, Takano K, Kanaya S Ref: Applied Environmental Microbiology, 78:1556, 2012 : PubMed
The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50 degrees C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70 degrees C and 7 min at 80 degrees C. LC-cutinase* had an ability to degrade poly(epsilon-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/mukat of pNP-butyrate-degrading activity) at pH 8.0 and 50 degrees C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.
        
Representative scheme of Polyesterase-lipase-cutinase structure and an image from PDBsum server
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