Characterization and mutagenesis of a two-component monooxygenase involved in para-nitrophenol degradation by an Arthrobacter strain

para-Nitrophenol (PNP) degradation cluster was cloned from a newly isolated Arthrobacter sp. strain NyZ415 by genome walking extending from a conserved region of gene encoding the oxygenase component of PNP monooxygenase. Sequence analysis indicated it contained 12 open reading frames including genes npdA1 and npdA2 which encoded the reductase and oxygenase components of PNP monooxygenase, respectively. Both NpdA1 and NpdA2 were expressed and purified to homogeneity. Hydroquinone and hydroxyquinol were captured when PNP was transformed by purified NpdA1A2 and para-benzoquinone was also captured for the first time as one of the products of PNP transformation by monooxygenase involved in the hydroxyquinol pathway. Sequence analysis showed four conserved residues (Arg100, Gln158, Arg161 and Thr193) related to the binding between the oxygenase component and substrate. Substitutions of any of above four residues to Ala have resulted in the complete loss of its activity on PNP transformation, indicating these four residues play important roles in the catalytic activity of PNP monooxygenase.