Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5

6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enz yme from Pseudomonas fluorescens TN5 was purified and characterized. The en zyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and dep ends on O-2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxy pyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer thera py. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotn ic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreemen t with the enzyme molecular mass, amino acid sequence of an internal peptid e, contains a putative FAD-binding site and is homologous to similar flavop roteins such as salicylate 1-monoxygenase.