Structure/function studies on engineered mutants of catechol 1,2-dioxygenase: experimental vs in silico approach.

Structure/function studies on engineered mutants of catechol 1,2-dioxygenase: experimental vs in silico approach. Valuable mutants of catechol 1,2-dioxygenase from Acinetobacter radioresistens S13 were produced with improved clorocatechols turnover and a change in specifity(1). The aim of the present project is to find a correlation beetwen the experimental data on the mutant activity and in silico calculations in order to predict further mutations that would improve pollutants recognition. The temperature-dependent activity increase was analysed in all variants in order to determine the activation free energy calculated on the basis of the kcat. The Arrhenius plot thus obtained describes the free energy of transition Ea from the ES to the ES* state. A peculiar aspect is that the lowering of the activation energy follows precisely the enlargements of the active site pocket: by comparing the energy required for the transition, using catechol as a substrate, it was observed that the value is higher for the WT than for the mutants (L69A, L69GA72G, A72G) except in the A72S mutant. The A72S Arrhenius plot showed a biphasic behavior, with Ea = 7680 cal/mol (between 10°C and 27°C) and Ea = 763 cal/mol (between 30°C and 37°C) (1). In the range of lower temperature, the value for A72S is consistently identical to the WT value. This is in agreement with the fact that no enlargement of the active site is expected by mutating an alanine to a serine. In the higher temperature range, instead, A72S showed a net decrease in Ea value, as if the volume had increased. An important detail is that, among all the obtained mutations, the serine is the only residue that could make an additional hydrogen bond. Waiting for native and mutants crystallographic structure (underway), a first in silico prediction was made to check if this property can be rationalised on WT and A72S models obtained by Modeller. These showed two hydrogen bond patterns around the serine 72. In the first one, the serine made only an hydrogen bond with glycine 75 through the carbossilic group of the peptide bond (as in the WT model); in another cluster, an additional hydrogen bond occurred between the oxydril hydrogen of the serine 72 and oxygen of C1 carbossil group of glutamic acid 77 or alanine 249 or leucine 69. In conclusion, an hypotetical explanation of biphasicity is related to the possibility that the serine makes an additional hydrogen bond when temperature increases: in this case, the oxydril group could switch; as consequence, the cavity volume could enlarge and the Ea decreases. As the serine, also threonine could be a hydrogen bonds donor/acceptor. Recently the A72T mutant was obtained: the first experimental data seem to confirm A72S data, since also A72T Arrenhius plot showed a biphasic behaviour. References: (1) R. Caglio, F. Valetti*, P. Caposio, G. Gribaudo, E. Pessione, C. Giunta, "Fine tuning of catalytic properties of catechol 1,2 dioxygenase by active site tailoring" ChemBioChem 2009 (in press).