GAIN-OF-FUNCTION MUTANTS OF CATECHOL 1,2 DIOXIGENASE FOR CATALYSIS ON CHLORINATED ALTERNATIVE SUBSTRATES

In order to increase the range of substrates recognized by catechol 1,2-dioxygenase IsoB from A. radioresistens S13 (Di Nardo et al., 2004; Caposio et al., 2002; Pessione et al., 2001), a double mutant L69G A72G a mutant L69A were created by rational design and other 6 active mutants (A72S, A72T, A72G, A72P, A72D and A72N) were obtained by applying site-saturation mutagenesis by PCR methods and Quikchange kit. The activity of the purified mutants was assayed to determinate the kinetic constants for the natural substrate, methylcatechols, chlorocatechols and then tested on halogenated catechols and other substituted diols of environmental interest to select for gain-of-function variants. 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. A low Km was measured also on 3-methyl and 4-chloro catechol. L69G A72G displayed a highly enhanced preference for catechols with substituents in 3- and 4- compared to catechol. Both L69G A72G and L69A are gain-of-function variants for recognition and aromatic ring cleavage of p-tert-butylcatechol (CAS No. 98-29-3) with kcat of 6 and 8 min-1, and of 4,5-dichlorocatechol (CAS No. 3428-24-8) with kcat of 14.5 and 13 min-1. Also mutations A72G, A72D and A72N convey the novel activity on 4,5-dichlorocatechol with kcat of 18.2, 22.4 and 6.3 min-1 respectively. A72G presents an increase of activity on 4-chloro catechol, with substrate preference comparable to the double mutant L69G A72G, but higher specific activity. A72D, in spite the similarity of the substitution with the active site of a chlorocatechol dioxygenase (Ferraroni et al., 2004), displays a general increase of Km values and very similar kcat towards both catechol and methyl or chloro derivatives. The most interesting mutant for improved recognition of chlorinated catechols seems to be the A72N, with a level of catalysis of 4-chlocatechol that is higher than on catechol, completely inverting the preference of WT enzyme. Only one inactive mutant isolated from the random mutagenesis of codon 72 namely A72K, bear amino acid substitution that can be reasonably considered not compatible, we suppose, for size and charge, with the active site cavity. The effect of the active site pocket mutations on catalysis were rationalised in term of structure-function interplay on the basis of structural alignment and active site modelling, using as a template the crystallographic structure of C1,2O from A. calcoaceticus ADP1 (Vetting and Ohlendorf, 2000). Crystallisation studies and silica gel entrapment experiments are underway in order to confirm the hypothesis and collect further data for theoretical studies and bio catalysis applications of the obtained variants.