Pendimethalin Nitroreductase Is Responsible for the Initial Pendimethalin Degradation Step in Bacillus subtilis Y3

Abstract

Pendimethalin [N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a selective preemergence dinitroaniline herbicide. Several fungi and bacteria have been reported to degrade pendimethalin, but the enzymes or genes involved in this process have not been characterized. Nitroreduction is the initial degradation and detoxification step for pendimethalin. In this study, a pendimethalin nitroreductase (PNR), responsible for the nitroreduction of pendimethalin, was purified from the pendimethalin-degrading strain Bacillus subtilis Y3. Based on a comparison of its mass fingerprints with all of the deduced proteins from the draft genome of strain Y3, a protein annotated as a nitroreductase was identified, and its corresponding encoding gene was termed pnr. PNR was a functional homodimer with a subunit molecular mass of approximately 23 kDa. PNR reduced the C-6 nitro group of the aromatic ring of pendimethalin, yielding 2-nitro-6-amino-N-(1-ethylpropyl)-3,4-xylidine. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. However, the number of reduced nitro groups was two instead of one, which differed from the nitroreduction of pendimethalin by PNR and which may be due to the symmetry in the chemical structures of the two nitro groups. A detoxification assay revealed that 2-nitro-6-amino-N-(1-ethylpropyl)-3,4-xylidine (PNR-reduced pendimethalin) showed no inhibitory effect on the growth of Saccharomyces cerevisiae BY4741, whereas pendimethalin showed an obvious inhibitory effect on its growth, indicating the detoxification effect of pendimethalin by PNR. Therefore, PNR has potential in pendimethalin detoxification applications. This report describes an enzyme (and corresponding gene) involved in the biodegradation of pendimethalin and dinitroaniline herbicides. N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a widely used selective preemergence dinitroaniline herbicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency (EPA) has classified pendimethalin as a persistent bioaccumulative toxin. To date, no enzymes or genes involved in pendimethalin biodegradation have been reported. In the present study, the gene pnr, which encodes the nitroreductase PNR, responsible for the nitroreduction of pendimethalin, was cloned from the pendimethalin-degrading strain Bacillus subtilis Y3. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. The reduction of pendimethalin by PNR might eliminate its toxicity against Saccharomyces cerevisiae BY4741, indicating the application potential of PNR in the detoxification of pendimethalin. IMPORTANCE Pendimethalin [

Structure and substrate specificity determinants of NfnB, a dinitroaniline herbicide–catabolizing nitroreductase from Sphingopyxis sp. strain HMH

“…These characteristics of NfnB are different from those of PNR ( 17 ), the enzyme efficiency of which is similar among butralin, pendimethalin, oryzalin, and trifluralin. PNR also has higher efficiency values for these substrates (10.9–11.9 μM −1 s −1 ) than NfnB for butralin (1.6 μM −1 s −1 ).…”

“…Recently, two bacterial nitroreductases have been shown to metabolize dinitroaniline herbicides ( Fig. 1 A ): pendimethalin nitroreductase (PNR) from Bacillus subtilis Y3 ( 16 , 17 ) and NfnB from Sphingopyxis sp. strain HMH ( 18 ).…”

“…PNR and NfnB are oxygen-insensitive type I bacterial nitroreductases, with a low sequence identity of ∼18%, and are classified into the NfsB and PnbA subgroups, respectively. PNR, which employs both NADH and NADPH, is applicable for the nitroreduction of a broad range of dinitroaniline herbicides, with similar enzyme efficiency ( k cat / K m ) ( 16 , 17 ). Specifically, PNR nitroreduction was identified only at the C6-nitro group of pendimethalin, but both at nitro groups at C2 and C6 of butralin, oryzalin, and isopropalin ( Fig.…”

Gut microbial community response to herbicide exposure in a ground beetle

“…The nitroreduction has been indicated as the initial degradation and detoxification step for pendimethalin (Ni et al. 2016). Numerous pendimethalin‐degrading microorganisms have been isolated in the environment (Elsayed & El‐Nady, 2013; Strandberg & Scott‐Fordsmand, 2004) including Paracoccus sp.…”

Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001

“…The basis of the biotransformation is that NTRs catalyze the sequential transfer of two electrons from a cofactor to nitro groups in a variety of substrates to produce the nitroso, hydroxylamine, and/or amine derivatives . Nitroreduction is the quintessential first step in the biotransformation of various nitroaromatic and nitroheterocyclic compounds , and such reduction properties make NTRs the key factors in biodegradation of toxic nitro compounds , activation of nitro prodrugs , and biosynthesis of novel hydroxylamines and/or amines .…”