The gene encoding m-hydroxybenzoate hydroxylase (rnobA) was cloned from Comamonas testosteroni GZ39. MobA converts m-hydroxybenzoate and to a lesser extent p-hydroxybenzoate to protocatechuate. To explore the structural and functional relationships in phenolic acid monooxygenases, MobA was subjected to in vitro mutagenesis by error-prone PCR and the mutant MobAs were screened for their ability to oxidize phenol or 3-aminophenol. A mutant MobA with a single V257A substitution was able to transform phenol to catechol, providing the first example of monooxygenase acting on phenolic acids that can also hydroxylate phenol. The mutant MobA also has enhanced ability to transform resorcinol, hydroquinone, p-hydroxybenzoate, 2,5-dihydroxybenzoate, 3,4-dihydroxybenzoate, 3-chlorophenol, 4-chlorophenol, 4-chlororesorcinol, and 4-nitrophenol. Several MobA mutants were obtained for their ability to transform 3-aminophenol to a related substituted catechol. Mutant MobAs with single amino acid substitutions (H135P, A400G, or D416A) were derived from these mutants and verified for their ability to transform 3-aminophenol. (C) 2008 Elsevier Inc. All rights reserved.