Difference in kinetic behaviour of catechol 2,3-dioxygenase variants from a polluted environment

Abstract

In a previous environmental survey of a polluted area, the authors identified two catechol 2,3-dioxygenase (C23O) sequences predominant in environmental bacterial isolates mineralizing benzene and/or toluene and also in soil DNA extracts. In the present study, using information of stable operon arrangement and conserved gene sequences, the complete C23O ORFs of these two variants were cloned, sequenced and overexpressed. The variants differ in six nucleotide positions, and the putative protein sequences differ only by a single amino acid, Tyr or His, at position 218. Even though the three-dimensional model does not suggest a significant influence of such an amino acid substitution on enzyme function, the Tyr218 variant differed significantly from the His218 variant in lower turnover number and in lower apparent K m for catecholic substrates. These results are evidence of the importance for enzyme function of amino acids not directly influencing active site structure and prove the utility of recovering polymorphisms evolved and selected for special functions in natural ecosystems.

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

“…V max , k cat , and apparent K m values of C12O's with catechols were determined using 1 to 100 M substrate in air-saturated buffer, and kinetic data were calculated from the initial velocities using the MichaelisMenten equation by nonlinear regression (KaleidaGraph; Synergy Software). Since very low K m values were indicated by this method, kinetic data were finally determined from progress curves obtained from reactions with initial substrate concentrations of 10 M, as previously described for determination of low K m values of catechol 2,3-dioxygenases (20). The data sets obtained were fit to the Michaelis-Menten equation by nonlinear regression (KaleidaGraph; Synergy Software).…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a Bio-Rad Miniprotein II apparatus as previously described (26) (20) and scanned using a Fujifilm LAS-1000 charge-coupled device camera. The fluorescence intensity was integrated, and the relative intensities of the bands corresponding to C12O catA , C12O salD , MCI catB , and SalOH were determined using the AIDA 2.1 software package (Raytest Isotopenmessgeräte GmbH).…”

Degradation of Benzene by Pseudomonas veronii 1YdBTEX2 and 1YB2 Is Catalyzed by Enzymes Encoded in Distinct Catabolism Gene Clusters

“…In the well-characterized isopropylbenzene-degradative pathways of Pseudomonas putida RE204 and Pseudomonas fluorescens IP01 (13,14), the genes coding for the oxygenase subunits are organized in operons together with genes encoding a cis-dihydrodiol dehydrogenase and an extradiol dioxygenase of subfamily I.3.A (15). However, P. veronii 1YB2 and 1YdBTEX2 do not express a subfamily I.3.A extradiol dioxygenase (EXDO K2) during growth on benzene but recruit a subfamily I.2.A extradiol dioxygenase (catechol 2,3-dioxygenase [C23O]) (EXDO A) with high similarity to the archetype TOL plasmid-encoded enzyme (6,16). To our knowledge, such a recruitment has not been reported previously for the degradation of benzene or toluene via a dioxygenolytic route.…”

Degradation of 2,3-Dihydroxybenzoate by a Novel meta -Cleavage Pathway

“…2,3-Dihydroxy-p-cumate was a kind gift from Richard Eaton (18). 2-Hydroxy-3-carboxymuconic semialdehyde and 2-hydroxy-3-carboxy-6-oxo-7-methylocta-2,4-dienoate were prepared in situ by incubation of a solution containing 0.1 mM 2,3-dihydroxybenzoate or 2,3-dihydroxy-p-cumate in 50 mM phosphate buffer (pH 8.0) with cell extract of E. coli JM109(pGC23O) preinduced for the induction of DhbA, whereas HMS and HOPDA (0.1 mM) were prepared with cell extracts of E. coli JM109(pC23Ohis218) expressing catechol 2,3-dioxygenase (32).…”