The catechol 2,3-dioxygenase (C23O) encoded by the Pseudomonas putida xylE gene was over-produced in Escherichia coli and purified to homogeneity. The activity of the C23O required the reduced form of the Fe(II) ion since the enzyme was highly susceptible to inactivation with hydrogen perocide but reactivated with the addition of ferrous sulfate in conjunction with ascorbic acid. The C23O activity was abolished by treatment with the chemical reagents, diethyl-pyrocarbonate (DEPC), tetranitromethane (TNM), and 1-cyclohexy1-3-(2-morpholinoethyl) car-bodiimidemetho-ρ-toluenesulfontate (CMC), which are modifying reagents of histidine, tyrosine and glutamic acid, respectively. These results suggest that histidine, tyrosine and glutamic acid residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues may be good active sites for the enzyme activity. These amino acid residues are conserved residues among several extradion dioxygenases and have the chemical potential to serveas ligands for Fe(II) coordination. Analysis of random point mutants in the C23O gene derived by PCR technique revealed that the mutated positions of two mutants, T179S and S211R, were located near the conserved His165 amd Hos217 residues, respectively. This finding indicates that these two positions, along with the conserved histidine residues, are specially effective regions for the enzyme function.
Essential amino acids involved in the catalytic mechanisms of cytosine deaminase from Aspergillus fumigatus IFO 5840 were determined by chemical modification studies. The enzyme was perfectly inhibited by N-bromosuccinimide, chloramines-T, pyridoxal-5’-phosphate, and p-chloromercuribenzoate. It was strongly inhibited by phenylmethyl sulfonyl fluoride, and weakly inhibited by phenylglyoxal. The inactivation of the enzyme activity by p-CMB was reversed by sulfhydryl reagents. Furthermore, activities inhibited by chloramines-T, pyridoxal-5’-phosphate, results, we speculate that tryptophan, methionine, lysine and cysteine residues are located in ornear the active center of the cytosine deaminase, while a serine is indirectly involved I the enzyme activity.