Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis

The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strai n BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring clea vage mechanism. In an effort to improve the efficiency of this reaction, bp hC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Varian t enzymes containing single substitutions were constructed, and the amino a cid substitutions responsible for altered enzyme properties were identified . One variant enzyme, which contained an exchanged amino acid in the C-term inal part, revealed a higher level of stability during conversion of 3-chlo rocatechol than the wild-type enzyme. Two other variant enzymes contained a mino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a signif icantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermor e, these variant enzymes showed the novel capability to oxidize 3-methylcat echol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.