Affiliation:
1. Shandong Provincial Key Laboratory of Applied Mycology, College of Life Sciences, Qingdao Agricultural University, Qingdao, People's Republic of China
Abstract
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.
Funder
Chinese National Natural Science Foundation
Shandong province Natural Science Foundation
Qingdao Agricultural University Scientific Research Foundation
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
3 articles.
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