Novel Gene Encoding 5-Aminosalicylate 1,2-Dioxygenase from Comamonas sp. Strain QT12 and Catalytic Properties of the Purified Enzyme

Abstract

The 1,125-bp mabB gene encoding 5-aminosalicylate (5ASA) 1,2-dioxygenase, a nonheme iron dioxygenase in the bicupin family that catalyzes the cleavage of the 5ASA aromatic ring to form cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate in the biodegradation of 3-aminobenzoate, was cloned from Comamonas sp. strain QT12 and characterized. The deduced amino acid sequence of the enzyme has low sequence identity with that of other reported ring-cleaving dioxygenases. MabB was heterologously expressed in Escherichia coli cells and purified as a His-tagged enzyme. The optimum pH and temperature for MabB are 8.0 and 10°C, respectively. Fe^II is required for the catalytic activity of the purified enzyme. The apparent K_m and V_max values of MabB for 5ASA are 52.0 ± 5.6 μM and 850 ± 33.2 U/mg, respectively. The two oxygen atoms incorporated into the product of the MabB-catalyzed reaction are both from the dioxygen molecule. Both 5ASA and gentisate could be converted by MabB; however, the catalytic efficiency of MabB for 5ASA was much higher (∼70-fold) than that for gentisate. The mabB-disrupted mutant lost the ability to grow on 3-aminobenzoate, and mabB expression was higher when strain QT12 was cultivated in the presence of 3-aminobenzoate. Thus, 5ASA is the physiological substrate of MabB.IMPORTANCE For several decades, 5-aminosalicylate (5ASA) has been advocated as the drug mesalazine to treat human inflammatory bowel disease and considered the key intermediate in the xenobiotic degradation of many aromatic organic pollutants. 5ASA biotransformation research will help us elucidate the microbial degradation of these pollutants. Most studies have reported that gentisate 1,2-dioxygenases (GDOs) can convert 5ASA with significantly high activity; however, the catalytic efficiency of these enzymes for gentisate is much higher than that for 5ASA. This study showed that MabB can convert 5ASA to cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, incorporating two oxygen atoms from the dioxygen molecule into the product. Unlike GDOs, MabB uses 5ASA instead of gentisate as the primary substrate. mabB is the first reported 5-aminosalicylate 1,2-dioxygenase gene.

Keywords: 3-aminobenzoate; 5-aminosalicylate; Comamonas; biodegradation; dioxygenase.

FIG 1

Phylogenetic tree of Comamonas sp. QT12 in relation to other type strains within the Comamonas family based on the 16S rRNA gene sequences. GenBank accession numbers are indicated in parentheses. Sequence alignment was performed using ClustalW, and phylogenetic inferences were made by MEGA 6.0 software using neighbor-joining methods. Bootstrap values (from 1,000 replicates) are shown at nodes. Bar, evolutionary distance of 5 replacements per 1,000 nucleotide positions.

FIG 2

3-Aminobenzoate degradation in Comamonas sp. QT12. (A) HPLC analysis of 3-aminobenzoate biotransformation by resting cells of QT12. The signal was monitored at 210 nm. (B) Proposed pathway for the degradation of 3-aminobenzoate in strain QT12.

FIG 3

Phylogenetic analysis of MabB. The tree was constructed for MabB and several orthologous representative of dioxygenases of the bicupin family by using the neighbor-joining methods with a bootstrap of 1,000. The lengths of the lines are proportional to the genetic distance between proteins. The bar represents 0.2 amino acid substitution per site. 5NSADO, 5-nitrosalicylic acid 1,2-dioxygenase. GenBank accession numbers or protein identifications are listed at the end of each name.

FIG 4

Identification of the mabB gene in Comamonas sp. QT12. (A) RT-PCR analysis of mabB expression with cDNA (lane 1) or RNA (lane 1R) from cells in the presence of 3-aminobenzoate, cDNA (lane 2) or RNA (lane 2R) from cells in the absence of 3-aminobenzoate, genomic DNA (lane +), and ddH₂O (lane −) as the templates. (B) The expression level of mabB in the presence (bar 1) or absence (bar 2) of 3-aminobenzoate. Each value is the mean from three parallel replicates ± SD. (C) Construction of mabB-disrupted mutant strain Comamonas sp. QT12 ΔmabB. (D) Strain QT12 and mabB-disrupted mutant grown on a solid MSM plate containing 3-aminobenzoate as the sole carbon and nitrogen source.

FIG 5

Characterization of MabB. (A) SDS-PAGE of purified MabB. Lanes: M, protein markers (in kilodaltons); 1, MabB. (B) Spectrophotometric changes during the transformation of 5ASA by MabB. The reactions were initiated by the addition of 100 μM 5ASA and MabB, and the spectra were recorded every 1 min. Arrows indicate the direction of spectral changes. (C) pH optimization of MabB. (D) Temperature-dependent enzyme activity of MabB. Each value is the mean from three parallel replicates ± SD.

FIG 6

HPLC and ESI-MS analysis of MabB-catalyzing reactions. (A and B) HPLC analysis of the reactions of MabB at 0 min (A) and 20 min (B). (C) ESI-MS analysis of MabB catalyzing reaction; 200 μM 5ASA was mixed with MabB in 20 mM phosphate buffer (pH 8.0). After 20 min, 2 volumes of ethanol were added to the mixture to terminate the reaction. The product cis-ACOHDA is indicated. (D) ESI-MS analysis of ¹⁸O-labeling MabB-catalyzing experiments. The ¹⁸O-labeled cis-ACOHDA is indicated.

FIG 7

Analysis of MabB-catalyzing reaction with gentisate as the substrate. (A) Spectrophotometric changed during the transformation of gentisate by MabB. The spectra were recorded every 2 min. Arrows indicate the direction of spectral changes. (B) HPLC analysis of gentisate conversion by MabB.