A novel gene, encoding 3-aminobenzoate 6-monooxygenase, involved in 3-aminobenzoate degradation in Comamonas sp. strain QT12.

The biodegradation pathway of 3-aminobenzoate has been documented, but little is known about the sequence and biochemical properties of the proteins involved. In the present study, a 10,083-bp DNA fragment involved in 3-aminobenzoate degradation was identified in 3-aminobenzoate-degrading Comamonas sp. strain QT12. The mabA gene, whose encoded protein shares 39% amino acid sequence identity with 3-hydroxybenzoate 6-hydroxylase of Polaromonas naphthalenivorans CJ2, was identified on this DNA fragment, and the mabA-disrupted mutant was unable to grow on and convert 3-aminobenzoate. MabA was heterologously expressed in Escherichia coli and purified to homogeneity as an approximately ~ 48-kDa His-tagged protein. It was characterized as 3-aminobenzoate 6-hydroxylase capable of catalyzing the conversion of 3-aminobenzoate to 5-aminosalicylate, incorporating one oxygen atom from dioxygen into the product. It contains a non-covalent but tightly bound FAD as the prosthetic group and NADH as an external electron donor. 5-Aminosalicylate was produced with equimolar consumption of NADH. The apparent Km and kcat values of the purified enzyme for 3-aminobenzoate were 158.51 ± 4.74 μM and 6.49 ± 0.17 s-1, respectively, and those for NADH were 189.85 ± 55.70 μM and 7.41 ± 1.39 s-1, respectively. The results suggest that mabA is essential for 3-aminobenzoate degradation in strain QT12, and that 3-aminobenzoate is the primary and physiological substrate of MabA.