Protein engineering was applied on catechol 1,2-dioxygenase from A. radioresistens S13 (1,2) to produce active site mutants. Homology modelling on an available template (3) was applied to select mutation sites and the experimental data rationalised in terms of structure-function interplay. The activity of the mutants was assayed to determinate the kinetic constants for the natural substrate, methylcatechols, and then tested on chlorinated catechols of environmental interest to select for gain-of-function variants. Novel activity was observed towards 4,5 dichlorocatechol and 4-tert butyl catechol. Steady-state kinetic studies demonstrated that the double mutant L69G A72G and the mutant L69A have a 5 times lower Km than WT on 4-methyl catechol. A72G presents an increase of activity on 4-chlorocatechol. A72D, inspite the similarity of the substitution with the active site of a chlorocatechol dioxygenase (4) displays a general increase of Km values. A72N mutation completely inverts the preference of WT enzyme, having higher turnover on 4-chlocatechol that on catechol. REFERENCES 1.Di Nardo G. et al., Arch Biochem Biophys. 2004 Nov 1;431(1):79-87. 2.Pessione E. et al., Biol Chem. 2001 Aug;382(8):1253-61. 3.Vetting MW, Ohlendorf DH. Structure Fold Des. 2000;8(4):429-40. 4.Ferraroni M. et al., J Biol Chem. 2004; 279(26): 27646-55.

ACTIVE SITE ENGINEERING OF CATECHOL 1,2 DIOXYGENASE FOR CHLORINATED CATECHOLS RING CLEAVAGE

CAGLIO, Raffaella;VALETTI, Francesca;GIUNTA, Carlo
2007

Abstract

Protein engineering was applied on catechol 1,2-dioxygenase from A. radioresistens S13 (1,2) to produce active site mutants. Homology modelling on an available template (3) was applied to select mutation sites and the experimental data rationalised in terms of structure-function interplay. The activity of the mutants was assayed to determinate the kinetic constants for the natural substrate, methylcatechols, and then tested on chlorinated catechols of environmental interest to select for gain-of-function variants. Novel activity was observed towards 4,5 dichlorocatechol and 4-tert butyl catechol. Steady-state kinetic studies demonstrated that the double mutant L69G A72G and the mutant L69A have a 5 times lower Km than WT on 4-methyl catechol. A72G presents an increase of activity on 4-chlorocatechol. A72D, inspite the similarity of the substitution with the active site of a chlorocatechol dioxygenase (4) displays a general increase of Km values. A72N mutation completely inverts the preference of WT enzyme, having higher turnover on 4-chlocatechol that on catechol. REFERENCES 1.Di Nardo G. et al., Arch Biochem Biophys. 2004 Nov 1;431(1):79-87. 2.Pessione E. et al., Biol Chem. 2001 Aug;382(8):1253-61. 3.Vetting MW, Ohlendorf DH. Structure Fold Des. 2000;8(4):429-40. 4.Ferraroni M. et al., J Biol Chem. 2004; 279(26): 27646-55.
41st IUPAC World Chemistry Congress
Torino
5-11 agosto 2007
41st IUPAC World Chemistry Congress -Chemistry Protecting Health, Natural Environment and Cultural Heritage
-
232
232
protein engineering; bioremediation; oxygenase; chlorinated pollutants
R. Caglio; F. Valetti; C. Giunta
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/47088
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