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Agricultural and Environmental Chemistry

Biodegradation of the insecticide flonicamid by Alcaligenes faecalis CGMCC 17553 via hydrolysis and hydration pathways mediated by nitrilase Wen-Long Yang, Lei-Lei Guo, Zhi-Ling Dai, Ruo-Chen Qin, Yun-Xiu Zhao, and Yijun Dai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04245 • Publication Date (Web): 16 Aug 2019 Downloaded from pubs.acs.org on August 18, 2019

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Journal of Agricultural and Food Chemistry

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Biodegradation of the insecticide flonicamid by Alcaligenes

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faecalis CGMCC 17553 via hydrolysis and hydration pathways

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mediated by nitrilase

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Wen-Long Yang, Lei-Lei Guo, Zhi-Ling Dai, Ruo-Chen Qin, Yun-Xiu Zhao, Yi-

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Jun Dai*

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Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and

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Technology Research Center for Industrialization of Microbial Resources, College of Life

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Science, Nanjing Normal University, Nanjing 210023, People’s Republic of China

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*Address correspondence to: Yi-Jun Dai, tel: 86-25-85891731, fax: 86-25-85891067, e-mail:

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[email protected]

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ACS Paragon Plus Environment


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Abstract: Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO), a novel selective

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systemic pyridinecarboxamide insecticide, effectively controls hemipterous pests. However,

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microbial degradation of flonicamid, along with the enzymatic mechanism, has not been studied.

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Here, bacterial isolate PG13, which converts flonicamid into 4-(trifluoromethyl) nicotinol glycine

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(TFNG) and N-(4-trifluoromethylnicotinoyl) glycinamide (TFNG-AM), was isolated and

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identified as Alcaligenes faecalis CGMCC 17553. The genome of CGMCC 17553 contained five

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nitrilases but no nitrile hydratase, and recombinant Escherichia coli strains harboring CGMCC

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17553 nitrilase genes nitA or nitD acquired the ability to degrade flonicamid. Purified NitA

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catalyzed flonicamid into both TFNG and TFNG-AM, indicating dual functionality, while NitD

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could only produce TFNG-AM. Three-dimensional homology modeling revealed that aromatic

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amino acid residues in the catalytic pocket affected nitrilase activity. These findings further our

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understanding of the enzymatic mechanism of flonicamid metabolism in the environment and may

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help develop a potential bioremediation agent for the elimination of flonicamid contamination.

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Keywords: insecticide; biodegradation; Alcaligenes faecalis; flonicamid; nitrilase

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Introduction

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Flonicamid (N-cyanomethyl-4-trifluoromethylnicotinamide, FLO), a pyridinecarboxamide

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insecticide, demonstrates highly selective activity against pests belonging to the order Hemiptera,

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including cotton leafhopper, aphids, and mosquitoes.1-3 As such, flonicamid is commonly used to

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control pests on a variety of crops, such as hops, cabbage, cotton, wheat, and potatoes.4-7

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Mechanistically, flonicamid has been shown to block A-type potassium channels,8 causing

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toxicity by inhibiting inward rectifier K+ (Kir) channels of insect pests and disrupting salivary and

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renal excretion.9 Biological effects include loss of directed movement and suppression of feeding.

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Globally, neonicotinoids are the most commonly used class of insecticides; however, they pose

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a significant risk to wild bees and honeybees.10-12 Therefore, several countries have banned the

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use of neonicotinoid insecticides,13 necessitating the development of alternative pest control

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products. Compared with neonicotinoid insecticides, studies have shown that flonicamid is less

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toxic to bees, honeybees, and natural predators of insect pests; hence, it was recommended for

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widespread pest control.2, 7, 14, 15

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However, despite having decreased toxicity and being responsible for lower levels of residue

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build-up in the environment compared with neonicotinoid insecticides, flonicamid toxicity against

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predators of insect pests has been reported. For example, flonicamid was found to be toxic to

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coccinellid and chrysoperla, predators of pomegranate aphids,16 while a 17.39% mortality rate

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was observed in adult Coccophagus japonicus wasps following exposure to flonicamid.17

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flonicamid also had a sublethal impact on Nesidiocoris tenuis, a generalist predator of the

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Mediterranean Basin that is commonly used in biological control programs.18 Additionally, 3 / 43



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flonicamid reportedly reduced egg hatch numbers in Episyrphus balteatus19 and decreased the

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parasitism rates of Microplitis mediator and the parasitoid beetle Aleochara bilineata.20

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Flonicamid residue has also been detected in harvested food crops, including cabbages, bell

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peppers, and cucumbers,5, 21, 22 as well as in several watersheds around the Great Lakes Basin in

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the United States.23 Flonicamid and its metabolites, 5-trifluoromethylnicotinic acid (TFNA), 4-

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(trifluoromethyl) nicotinol glycine (TFNG), and N-(4-trifluoromethylnicotinoyl) glycinamide

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(TFNG-AM), were detected following application of the insecticide in orange groves in field

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studies,26 while flonicamid, TFNG, TFNA, and 4-trifluoromethylnicotinamide (TFNA-AM) were

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detected in dried hops.4 Even more worryingly, flonicamid residue has even detected in human

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serum and urine samples.24 This is of particular concern given that high doses of flonicamid

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caused DNA degradation and severe genome damage in mice.25

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With the increasing usage of flonicamid worldwide, environmental build-up of flonicamid

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residues seems more and more likely, which is a significant environmental concern. To remove

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flonicamid residues from the environment, photooxidation-based remediation methods have been

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trialed by several research groups. However, results were suboptimal when persulfate was added

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to the tested wastewater. The addition of ZnO- and TiO2-coated magnetic particles to the systems

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greatly enhanced the flonicamid degradation rate but was not economically viable.27,

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Microorganisms have been used as the basis of many insecticide degradation protocols that are

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considered fairly economical.29 However, there are currently no reports on the microbial

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degradation of flonicamid. Thus, in the current study, we intend to isolate flonicamid-degrading

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microorganisms from soil and characterize the underlying metabolic pathways and enzymes 4 / 43


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required for insecticide degradation. Our findings significantly enhance our understanding

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microbial flonicamid degradation in the environment and may help in the development of a novel

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bacterial-based method for flonicamid bioremediation.

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MATERIALS AND METHODS

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Chemicals and Media. Flonicamid (95% purity) was purchased from Hubei

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Zhengxingyuan Fine Chemical Co. (Wuhan, China) and TFNG was purchased from Sigma-

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Aldrich (St. Louis, MI). High-performance liquid chromatography (HPLC)-grade acetonitrile was

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purchased from Tedia Co. (Fairfield, OH). All other reagents were of analytical grade and were

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purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Mineral salt medium

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(MSM) used for screening and isolation of flonicamid-degrading microbes was prepared as

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described previously.30 Lysogeny broth (LB) contained 5 g of yeast extract, 10 g of tryptone, and

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10 g of NaCl in 1 L of deionized water (pH 7.2). LB agar medium was prepared by the addition

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of 20 g of agar to 1 L of liquid medium. The preparation of TFNG-AM is described in the

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Supporting Information.

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Isolation and Identification of Flonicamid-Degrading Bacterial Strain PG13.

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The method used to isolate flonicamid-degrading bacterial strains is outlined in the Supporting

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Information. Flonicamid-degrading isolates were identified by morphological analysis and 16S

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rRNA gene sequencing. Cell morphology was observed using an optical microscope after Gram

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staining. For all isolates, the 16S rRNA gene was amplified by colony polymerase chain reaction

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(PCR) using primers K1 (5ʹ-AACTGAAGAGTTTGATCCTGGCTC-3ʹ) and K2 (5ʹ-

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TACGGTTACCTTGTTACGACTT-3ʹ). The resulting amplicons were sequenced by Springern 5 / 43



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Co. (Nanjing, China). Nucleotide sequence data were deposited in the GenBank database. The

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16S rRNA gene sequence of PG13 was then compared against those already present in the

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GenBank database using the Basic Local Alignment Search Tool-nucleotide (BLASTn). A

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neighbor-joining phylogenetic tree for PG13 was then generated based on the 16S rRNA gene

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sequences using MEGA 6.

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Liquid Chromatography-Mass Spectrometry (LC-MS) Analyses. To identify

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the metabolites of flonicamid, flonicamid-transformed samples by PG13 were characterized by

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LC-MS analysis using an Agilent LC-MS system (model 1290 Infinity LC/6460 Triple Quad MS;

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Agilent, Santa Clara, CA) equipped with an electrospray ion source operated in both the positive

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ion mode and the negative ion mode. The detailed High-performance Liquid Chromatography

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(HPLC) Analysis condition was described in the Supporting Information.

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Biodegradation of Flonicamid in Soil. For soil biodegradation experiments, soil

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samples which was no pesticide application were collected from Nanjing, China. The

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physicochemical properties were measured in the Institute of Soil Science of the Chinese

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Academy of Sciences. The measurements were: pH 6.0, organic matter 17.24 g/kg, cation

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exchange capacity 21.61 cmol/kg. The soil sample was smashed and sieved by 0.55 mm sifter,

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then dry heat sterilization was practiced at 150℃ for 5 h. 10 g of sterilized dry soil samples were

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spread uniformly in 100-mL flasks. Bacteria pre-cultured in LB broth (30℃, 220 rpm for 18 h)

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were then collected and re-suspended in 200 mg/L sterilized FLO solution (50 mmol/L FLO in

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phosphate-buffered saline (PBS) [pH 7.0]) to an optical density at 600 nm (OD600) = 5 and aliquots

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(4 mL) of the bacterial suspension were then added to each soil sample immediately , resulting in 6 / 43


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a final FLO concentration of 80 mg/kg soil. For control, 4 mL of the bacterial suspension was

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instead by 4 mL sterilized FLO solution (50 mmol/L FLO in phosphate-buffered saline (PBS) [pH

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7.0]) added to each soil sample. The flasks were then incubated at 30°C and 35% humidity in the

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dark. Following incubation for the indicated time (0, 3, 6, 9 days), 12 mL of acetonitrile were

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added to each flask to examine FLO degradation. The flasks were then sealed and placed in a

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rotary shaker at 220 rpm for 2 h. Supernatants were collected following centrifugation at 10,000

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× g for 10 min and filtered prior to HPLC analysis described in the Supporting Information.

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Genomic DNA Sequencing and Cloning and Over-Expression of Nitrilase

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Genes in E. coli Rosetta (DE3). Total Genomic DNA was extracted from the flonicamid-

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degrading isolate using a MiniBEST Bacterial Genomic DNA Extraction Kit (TaKaRa, Dalian,

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China). Whole-genome sequencing and annotation was conducted by Beijing Genomics Institute

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Co. (Beijing, China). The primers used for amplification of nitrilase genes contained EcoRI and

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XhoI restriction enzyme sites are listed in Table 1. Primers were synthesized by Sangon Biotech

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Co. (Shanghai, China). Reaction mixtures for PCR analysis contained 1× PrimeSTAR Max

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Premix (TaKaRa), forward and reverse primers (1 mmol/L), 1 ng of DNA template, and ultrapure

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water to a final volume of 20 μL. Amplification was performed using a Bio-Rad Thermal Cycler

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(Bio-Rad, Hercules, CA) with the following program: hot start at 95°C for 5 min, followed by 31

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cycles of 95°C for 50 s, 54°C for 60 s, and 72°C for 60 s, and a final extension at 72°C for 10

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min. The resulting amplicons were verified by electrophoresis on 1% agarose gels in 1× TAE

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buffer followed by staining with ethidium bromide.

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Following verification, a ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., 7 / 43



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Nanjing, China) was used to recombine the EcoRI/XhoI-digested PCR products into expression

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vector pET28a as per the manufacturer’s protocols. The resulting recombinant plasmids

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containing nitrilase genes were individually transformed into competent E. coli Rosetta cells as

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described by Sun et al.31

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Enzymatic Activity Assay. Nitrilase gene expression was induced in the recombinant E.

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coli Rosetta (DE3) strains by the addition of 0.2 mmol/L isopropyl β-D-thiogalactoside (IPTG),

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0.2 mmol/L CoCl2 was added. Following incubation for 6 h at 30°C, sodium dodecyl sulphate

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polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess protein expression.

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Gels were stained with Coomassie Brilliant Blue. The activity of the expressed proteins against

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flonicamid was then examined as described in the resting cells biodegradation assays, with

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incubation at 30°C, 220 rpm, for 20 min. The culture supernatants were then collected and

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subjected to HPLC analysis described in the Supporting Information.

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Enzyme Purification and Biochemical Characterization. N-terminal 6× His-

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tagged NitA and NitD overexpressed in E. coli Rosetta (DE3) were purified by affinity

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chromatography as per the manufacturer’s instructions (Novagen, Madison, WI). SDS-PAGE was

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then performed to assess protein expression. Gels were stained with Coomassie Brilliant Blue.

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Nitrilase activity was determined by HPLC analysis condition was described in the Supporting

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Information. One unit (U) of enzyme activity was defined as the amount of enzyme required to

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catalyze the generation of 1 μmol of TFNG or TFNG-AM in 1 min. The optimal temperature for

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the degradation of flonicamid by NitA and NitD was determined by adding purified protein

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(concentration) to PBS (50 mmol/L, pH 7.0) containing 200 mg/L flonicamid and incubating the 8 / 43


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mixtures at temperatures ranging from 20–50°C (NitA) or 20–60°C (NitD) for 2 h. The optimal

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pH for the degradation of flonicamid by NitA and NitD was determined using the same system at

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30°C and 800 rpm for 2 h in PBS, citric acid/sodium citrate (CA/SC) or Tis/HCl, pH values

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ranging from 5.0–9.0. The same system was also used to examine the influence of various metal

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ions on flonicamid degradation by NitA and NitD. For these assays, PBS was supplemented with

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0.1 mmol/L EDTA, MgSO4, CoCl2, CaCl2, CuCl2, FeCl2, MnCl2, MoCl5, or ZnCl2.

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The kinetic parameters of NitA and NitD were examined in a 1-mL reaction mixture containing

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5 μL of purified NitA (2.75 mg/mL) and 995 μL of flonicamid (50–1000 mg/L) dissolved in PBS

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(50 mmol/L, pH 7.0). Mixtures were incubated at 40°C and 800 rpm for 30 min. The kinetic

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parameters of NitD were examined in a 1-mL reaction mixture containing 5 μL of purified NitD

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(1.09 mg/mL) and 995 μL of flonicamid (400–3000 mg/L) dissolved in PBS (50 mmol/L, pH 8.0).

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Mixtures were incubated at 40°C and 800 rpm for 10 h. The kinetic parameters of NitA and NitD

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were analyzed by Origin 8.5 nonlinear curve fit (MichaelisMenten).

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The substrate specificity of NitA and NitD was examined in a 1-mL reaction mixture containing

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20 μL of purified protein (NitA, 7.28 mg/mL; NitD, 11.73 mg/mL) and 980 μL of 200 mg/L

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flonicamid, acetamiprid (ACE), thiacloprid (THI), indolyl-3-acetonitrile (IAN), or 3-

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cyanopyridine (3-CP) dissolved in PBS (50 mmol/L, pH 7.0). Mixtures were incubated at 30°C

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and 800 rpm for 1 h (NitA) or 3 h (NitD). All the reactions were analyzed by HPLC analysis

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described in the Supporting Information.

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NitA Activity Against TFNG-AM. The enzymatic activity of NitA against TFNG-AM

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was examined in a 1-mL reaction mixture containing 30 μL of NitA (7.49 mg/mL) and 970 μL of 9 / 43



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TFNG-AM (250 mg/L) dissolved in PBS (50 mmol/L, pH 7.0). Mixtures were incubated at 30°C

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and 800 rpm for 30 min. The reaction was analyzed by HPLC analysis described in the Supporting

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Information.

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Homology Modeling of the Nitrilases. Three-dimensional homology models of the five

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nitrilases

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(https://swissmodel.expasy.org/interactive). The crystal structure of Nit6803 (PDB accession

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code 3WUY, x-ray diffraction resolution 3.10 Å) from Synechocystis sp. PCC6803 was used as

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template for NitA model construction. The crystal structure of N-carbamoylputrescine

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amidohydrolase from Medicago truncatula (PDB accession code 5H8I, x-ray diffraction

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resolution 1.97 Å) was used as a template for NitB model construction. The crystal structure of

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Nit2 from Saccharomyces cerevisiae strain ATCC 204508/S288c (PDB accession code 4H5U, x-

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ray diffraction resolution 1.92 Å) was used as template for NitC and NitE model construction.

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The crystal structure of PH0642 from Pyrococcus horikoshii (PDB accession code 1J31, x-ray

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diffraction resolution 1.60 Å) was used as template for NitD model construction. The qualities of

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the constructed nitrilase structures were then evaluated using GMQE32 and QMEAN33. The

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structures of the modeled nitrilases were analyzed by Chimera (www.cgl.ucsf.edu/ chimera),

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while active sites and catalytic triads within the nitrilases were predicted by BLAST sequence

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analysis against other nitrilase sequences in the Reference proteins (refseq_protein) database

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(Figures S7, S8, S9, S10, and S11 of the Supporting Information).

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RESULTS AND DISCUSSION

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were

developed

using

the

SWISS-MODEL

workspace

Isolation and Identification of Flonicamid-Degrading Microbes. A total of 15 10 / 43


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colonies showing obvious morphological differences on LB agar plates following culture in MSM

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broth containing 500 mg/L flonicamid were selected and tested for their ability to degrade

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flonicamid. Among these, one isolate, designated PG13, was confirmed to degrade flonicamid.

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Assays showed that PG13 could transform flonicamid into two products, referred to as P1 and P2

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(Figure 1A). PG13 formed yellow, wettish, roundish, convex colonies on LB agar, with

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morphological analysis showing that the isolate was a Gram negative, rod-shaped, non-spore-

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forming bacterium. Nucleotide BLAST analysis of the 16S rRNA gene (GenBank accession

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number MK888682) revealed that PG13 showed 96.83% nucleotide sequence identity to the

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corresponding gene from A. faecalis strain PNP6. The neighbor-joining phylogenetic tree

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generated by MEGA 6 based on 16S rRNA gene sequences showed that PG13 clustered with A.

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faecalis strain PNP6 but had more distant evolutionary relationships with other Alcaligenes

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species (Figure S1 in Supplementary Information). Therefore, PG13 was identified as A. faecalis

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and was deposited at the China General Microbiological Culture Collection Center (CGMCC)

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(Beijing, China) under accession number CGMCC 17553.

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Many other A. faecalis strains have demonstrated the ability to degrade organic pollutants,

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including phenanthrene, endosulfan, and nicosulfuron.34-36 Endosulfan is a chlorinated cyclodiene

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insecticide, while nicosulfuron is a sulfonylurea herbicide. However, this is the first report of the

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degradation of flonicamid, a cyano-group-containing insecticide, by an A. faecalis strain.

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Identification of the Metabolites. HPLC analysis showed that the retention times of

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products P1 and P2 were 3.5 and 4.5 min, respectively, which correspond to the retention times

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of TFNG and TFNG-AM, respectively. LC-MS analysis revealed that metabolite P1 exhibited 11 / 43



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peaks at 249, 271, 287, 519, 535, 247, and 495 m/z, corresponding to [M+H]+, [M+Na]+, [M+K]+,

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[2M+Na]+, [2M+K]+, [M−H]−, and [2M−H]−, respectively (Figure 1B and 1C). Metabolite P2 had

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peaks at 248, 270, 286, and 246 m/z, corresponding to [M+H]+, [M+Na]+, [M+K]+, and [M−H]−,

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respectively (Figure 1D and 1E). Therefore, the relative molecular weights of P1 and P2 were

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calculated as 248 and 247, respectively. Previous reports have shown that metabolic products of

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flonicamid, including TFNG, TFNG-AM, TFNA, and TFNA-AM, were present in crops

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following flonicamid application. The relative molecular weights of TFNG and TFNG-AM are

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248 and 247, respectively, which is consistent with the calculated molecular weights of P1 and

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P2, respectively (Figure 2A). These results therefore suggest that A. faecalis CGMCC 17553

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degrades flonicamid via hydrolysis and hydration pathways, two general chemical reaction

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pathways of cyano-group insecticides.

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Time Course of Flonicamid Degradation by Resting Cells and Degradation

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in Soil by A. faecalis CGMCC 17553. As shown in Figure 3A, resting A. faecalis CGMCC

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17553 cells reduced the initial concentration of flonicamid from 915 μmol/L to 11 μmol/L over

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96 h, corresponding to an overall degradation rate of 98.8%. During this period, 799 μmol/L

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TFNG and 101 μmol/L TFNG-AM were produced, resulting in a molar conversion rate of 99.5%.

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Overall, 88.3% and 11.2% of the substrate was transformed into TFNG and TFNG-AM,

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respectively. The half-life of flonicamid in the presence of A. faecalis CGMCC 17553 was 15 h.

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These results indicated that A. faecalis CGMCC 17553 efficiently degraded flonicamid to TFNG

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via hydrolysis, and that this is the main metabolic pathway of flonicamid degradation.

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Inoculation of A. faecalis CGMCC 17553 into soil resulted in a flonicamid degradation rate of 12 / 43


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85.0% within 9 days, with the half-life of flonicamid calculated as 3.3 days. Specifically, soil

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concentrations of flonicamid were decreased from 0.40 μmol/g of soil to 0.06 μmol/g of soil over

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the 9-day period. In the absence of A. faecalis CGMCC 17553 inoculation, 42.0% of the

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flonicamid was degraded in the same time period, corresponding to a half-life of 9.8 days (Figure

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4B). Therefore, inoculation of A. faecalis CGMCC 17553 into soil accelerated flonicamid

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degradation, indicating that microorganisms may be one of the major factors affecting flonicamid

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degradation in soil.

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A. faecalis CGMCC 17553 Genome Sequencing and Cloning and

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Overexpression of Nitrilase Genes in E. coli Rosetta (DE3). TFNG and TFNG-AM

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are the products of flonicamid hydrolysis and hydration pathways, respectively, which require the

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activity of nitrile hydratase/amidases or nitrilases.37 Analysis of the complete genome sequence

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of A. faecalis CGMCC 17553 revealed only five nitrilase-encoding genes: nitA (GenBank

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accession no. MK888683), nitB (GenBank accession no. MK926977), nitC (GenBank accession

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no. MK926978), nitD (GenBank accession no. MK926979), and nitE (GenBank accession no.

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MK926980). Interestingly, no nitrile hydratase-encoding genes were identified in the A. faecalis

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CGMCC 17553 genome. Nitrilases (EC 3.5.5.1) either hydrolyze nitriles into their corresponding

256

carboxylic acids or hydrate them into their corresponding carboxylic amides.38 The five nitrilase-

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encoding genes from A. faecalis CGMCC 17553 were amplified by PCR and ligated into

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expression vector pET28a. The recombinant plasmids were then transformed into E. coli Rosetta

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(DE3) and protein expression was induced. SDS-PAGE analysis showed that all of the target

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proteins were successfully expressed with the major soluble fractions (Figure 4A). E. coli Rosetta 13 / 43



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(DE3) overexpressing NitA degraded most of the available flonicamid into TFNG and TFNG-

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AM, while the NitD over-expressing strain only degraded 9.4% of the initial flonicamid, with

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only the TFNG-AM degradation product detected. Interestingly, E. coli Rosetta strains

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overexpressing NitB, NitC, or NitE showed no catalytic activity against flonicamid (Table 2).

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Results of SDS-PAGE analysis of the purified NitA and NitD proteins are shown in Figure 4B

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(lane 2) and Figure 4C (lane 2). Purified NitA degraded 77.9% of the starting concentration of

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flonicamid (200 mg/L) within 180 min, with 60.9% of the product identified as TFNG and the

268

remaining 30.1% identified as TFNG-AM (Figure 3C), suggesting that TFNG is the major

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degradation product. Purified NitD degraded 44.1% of the starting flonicamid concentration (200

270

mg/L) within the allotted 180 min (Figure 3D), but only produced TFNG-AM. Overall, the

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enzymatic behavior of the purified proteins was consistent with that of the A. faecalis CGMCC

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17553 cells.

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Enzymatic Characterization of NitA. The optimal pH for flonicamid degradation

274

by NitA was 7.0, with 100% enzyme activity observed at this pH. However, significant differences

275

in the activity of NitA against flonicamid were observed as the pH was adjusted between pH 5.0

276

and pH 9.0 (Figure 5A). At pH 5.0, enzyme activity was only 22.87% of that at the optimal pH,

277

but increased slightly when the pH was increased to 6.0. Activity again decreased when the pH

278

was adjust from pH 7.0 to pH 8.0, with another major drop in activity observed following an

279

increase to pH 9.0. Thus, NitA only exhibited activity within a narrow pH range. When NitA was

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preincubated at pH values ranging from 6.0–9.0, there was only a slight change in enzymatic

281

activity against flonicamid, with 100% activity observed at pH 8.0. In comparison, preincubation 14 / 43


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at pH 5.0 resulted in activity levels only 44.95% of that of the highest enzyme activity (Figure

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5B). Thus, NitA remained stable across a broad pH range.

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Maximum NitA activity (100%) was observed at 40°C. However, at 50°C, only 4.1% of the

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maximum activity remained, indicating that NitA cannot tolerate high temperatures (Figure 5C)

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and only exhibits activity within a narrow temperature range. When preincubated at temperatures

287

ranging from 30–50°C, the highest NitA activity against flonicamid was observed at 30°C, which

288

was defined as 100% activity. Activity levels decreased when temperatures were increased

289

between 30°C and 50°C, confirming that the enzyme was not stable at high temperatures (Figure

290

5D).

291

Co2+ had a positive effect on flonicamid degradation by NitA, with an observed 18.4%

292

increase in enzyme activity compared with the unsupplemented control. In comparison, Ca2+,

293

EDTA, and Fe2+ inhibited NitA enzyme activity by 7.2%, 14.3%, and 34.0%, respectively, while

294

Mg2+ and Mn2+ exhibited no effect on NitA enzyme activity. Importantly, Cu2+ and Zn2+

295

completely inhibited the ability of NitA to degrade flonicamid (Figure 5E). All of the tested

296

organic solvents inhibited NitA activity against flonicamid to some degree, with the observed

297

activity of NitA in the presence of ethyl alcohol, methyl alcohol, methylene dichloride, ethyl

298

acetate, and n-butyl alcohol decreased by 10.3%, 19.4%, 23.9%, 48.1%, and 24.8%, respectively

299

(Figure 5F).

300

Analysis of the kinetic parameters of the reaction revealed that NitA degradation of

301

flonicamid showed classic Michaelis-Menten kinetics. For the formation of TFNG, the Vmax was

302

1.18 U/mg and the Km was 1.62 mmol/L (Figure 6A), with corresponding values of 0.58 U/mg 15 / 43



303

and 0.59 mmol/L, respectively, for the formation of TFNG-AM (Figure 6B). Substrate specificity

304

assays showed that NitA had no activity against ACE, THI, IAN, or 3-CP, indicating that NitA

305

has a narrow substrate specificity (Figure 2B).

306

Enzymatic Characterization of NitD. The optimal pH for flonicamid degradation by

307

NitD was 8.0, with enzyme activity at pH 8.0 defined as 100%. As with NitA, the activity of NitD

308

against flonicamid decreased with alterations in the pH away from the optimum, with only 9.34%

309

and 57.10% of the highest enzyme activity remaining at pH 5.0 and pH 9.0, respectively (Figure

310

7A). Therefore, both NitA and NitB could be used to degrade flonicamid under neutral or slightly

311

alkaline pH conditions, although the activity of NitA is slightly higher. When NitD was

312

preincubated at pH values ranging from 5.0–9.0, there was little alteration in enzyme activity

313

against flonicamid, and the highest enzyme activity was observed at pH 8.0 (defined as 100%

314

enzyme activity) (Figure 7B). Thus, although decreased compared with optimal conditions, NitD

315

demonstrated activity across a broad range of pH conditions.

316

Maximum (100%) NitD enzyme activity against flonicamid was observed at 40°C, and large

317

alterations in activity were observed across the tested temperature range (20–70°C). At 70°C, only

318

7.5% of the maximum activity remained, indicating that NitD cannot tolerate high temperatures

319

(Figure 7C). When preincubated at temperatures ranging from 30–60°C, the highest (100%) NitD

320

activity against flonicamid was observed at 20°C. However, activity decreased as temperatures

321

increased from 20°C to 60°C, with only 1.6% of the highest activity remaining at 60°C. These

322

results confirmed that NitD does not tolerate high temperatures (Figure 7D).

323

EDTA, Ca2+, Co2+, Fe2+, Mg2+, and Mn2+ exhibited no effect on the activity of NitD, while Cu2+ 16 / 43


Page 16 of 43

Page 17 of 43


324

and Zn2+ inhibited NitD activity by 64.9% and 49.6%, respectively, compared with the control

325

(Figure 7E). While Cu2+ and Zn2+ strongly inhibited NitD enzyme activity, the inhibition was less

326

pronounced than that observed for NitA. Methylene dichloride enhanced NitD activity by 7.6%,

327

while ethyl alcohol, methyl alcohol, ethyl acetate, and n-butyl alcohol decreased the activity of

328

NitD by 3.5%, 5.8%, 12.7%, and 8.7%, respectively, compared with the control (Figure 7F).

329

Except for methylene dichloride, the levels of NitD inhibition by the organic solvents were similar

330

to results obtained for NitA.

331

Analysis of the kinetic parameters indicated that NitD degradation of flonicamid demonstrated

332

classic Michaelis-Menten kinetics. The Vmax of the NitD degradation reaction was 0.18 mU/mg,

333

while the Km was 145.87 mmol/L (Figure 6C). NitD demonstrated a reduced ability to form

334

TFNG-AM compared with NitA. Substrate specificity assays showed that NitD was not active

335

against ACE, THI, IAN, or 3-CP, displaying a similarly narrow substrate specificity to NitA.

336

NitA Activity Against TFNG-AM. Activity assays showed that NitA had no activity

337

against TFNG-AM. This indicated that TFNG and TFNG-AM were formed independently.

338

Therefore, we propose that flonicamid degradation by A. faecalis CGMCC 17553 is achieved

339

through the hydration of flonicamid into TFNG-AM by both NitA and NitD, and the hydrolysis

340

of flonicamid into TFNG by NitA only. NitA is thus a bifunctional enzyme.

341

Analysis of the Nitrilase Homology Models. Homology modeling of the nitrilases

342

from A. faecalis CGMCC 17553 was conducted using the SWISS-MODEL workspace (Figure

343

8). The homology model of NitA, which was generated based on the model crystal structure of

344

Nit6803, had a GMQE value of 0.62 and a QMEANA value of −2.71 (Figure 8A and Figure S2 17 / 43



345

of the Supporting Information). The amino acid sequence identity between NitA and Nit6803 was

346

35.2%. Based on alignments against selected nitrilase sequences, Glu-48, Lys-133, and Cys-167

347

were predicted to constitute the catalytic triad, while Glu-48, Lys-133, Cys-167, Thr-137, Glu-

348

140, Gly-168, Asn-170, Thr-171, and Tyr-191 were predicted to make up the putative active site

349

(Figure 9A and Figure S7 of the Supporting Information).

350

The homology model of NitB was generated based on the model crystal structure of M.

351

truncatula MtCPA and had a GMQE value of 0.51 and QMEANA value of −4.77 (Figure 8B and

352

Figure S3 of the Supporting Information). The sequence identity between NitB and Nit6803 was

353

19.58%. Alignment against selected nitrilase sequences revealed that Glu-47, Lys-129, and Cys-

354

163 made up the catalytic triad, while Glu-47, Lys-129, Thr-133, Cys-163, Trp-164, His-166,

355

Leu-167, and Trp-187 were predicted to form the putative active site (Figure 9B and Figure S8 of

356

the Supporting Information).

357

The homology model of NitC, generated based on the model crystal structure of S. cerevisiae

358

Nit2, had a GMQE value of 0.72 and a QMEANA value of −1.58 (Figure 8C and Figure S4 of the

359

Supporting Information). The sequence identity between NitB and Nit6803 was 35.97%.

360

Alignment against selected nitrilase sequences showed that the catalytic triad comprised Glu-42,

361

Lys-112, and Cys-149, while Glu-42, Lys-112, Tyr-116, Glu-123, Cys-149, Tyr-150, Leu-152,

362

Arg-153, and Ala-174 were predicted to form the putative active site (Figure 9C and Figure S9 of

363

the Supporting Information).

Page 18 of 43

364

The homology model of NitD was generated by SWISS-MODEL based on the model crystal

365

structure of hypothetical protein PH0642 (Figure 8D and Figure S5 of the Supporting Information), 18 / 43


Page 19 of 43


366

to which NitD showed 25.97% similarity. The GMQE value was 0.57 and the QMEANA value

367

was −3.38. Alignment against selected nitrilase sequences revealed that Glu-42, Lys-129, and

368

Cys-163 constituted the catalytic triad, while Glu-42, Lys-129, Thr-133, Glu-136, Cys-163, Trp-

369

164, Asn-166, Tyr-167, and Thr-187 were predicted to form the putative active site (Figure 9D

370

and Figure S10 of the Supporting Information).

371

The homology model of NitE was generated based on the model crystal structure of S.

372

cerevisiae Nit2, with a resulting GMQE value of 0.70 and a QMEANA value of −2.06 (Figure 8E

373

and Figure S6 of the Supporting Information). The sequence identity between NitB and Nit6803

374

was 33.08%. Alignment against selected nitrilase sequences showed that Glu-43, Lys-114, and

375

Cys-154 formed the predicted catalytic triad, while Glu-43, Asn-97, Lys-114, Tyr-118, Glu-129,

376

Cys-154, Tyr-155, Met-157, Arg-158, and Ala-179 made up the putative active site (Figure 9D

377

and Figure S11 of the Supporting Information).

378

While all five nitrilases showed a similar three-dimensional structure and a catalytic triad

379

comprising Glu, Lys, and Cys residues, their active sites were more varied. In NitA, the active

380

site amino acid residue adjacent to catalytic triad amino acid residue Cys-167 was Gly-168, which

381

has a hydrogen side chain, is non-polar, and occupies little space. In comparison, aromatic amino

382

acid residues Trp-164 (NitB), Tyr-116 and Tyr-150 (NitC), Trp-164 (NitD), and Tyr-118 and Tyr-

383

155 (NitE) were located in the corresponding positions in the catalytic pockets of the other

384

nitrilases. These residues occupy more space and may inhibit the binding of flonicamid within the

385

catalytic pocket, thereby inhibiting enzymatic activity against flonicamid. In addition, active site

386

residues Tyr-116 of NitC and Tyr-118 of NitE also occupied more space within the catalytic 19 / 43



387

pocket than the Thr-137 residue located at the corresponding position of NitA, any may therefore

388

also block the binding of flonicamid. The random coil of NitE at the catalytic pocket may also

389

take up space, further blocking the binding of flonicamid.

390

In conclusion, this is the first report of microbial degradation of flonicamid. A. faecalis strain

391

CGMCC 17553 was shown to efficiently transform flonicamid into TFNG and TFNG-AM. The

392

bifunctional nitrilase, NitA, of A. faecalis CGMCC 17553 was responsible for the transformation

393

of flonicamid into both TFNG and TFNG-AM, while nitrilase NitD only converted flonicamid

394

into TFNG. Aromatic amino acids within the catalytic pockets of several of the A. faecalis

395

CGMCC 17553 nitrilases may inhibit the flonicamid binding and therefore reduce nitrilase

396

activity. This study enhances our understanding of flonicamid degradation by microbes in the

397

environment and describes a novel bioremediation bacterium for the degradation of flonicamid

398

residues.

399

ASSOCIATED CONTENT

400

Supporting Information

401

The Supporting Information is available free of charge on the ACS Publications website.

402

Methods for the preparation of TFNG-AM and the isolation of bacteria from soil are provided

403

here. High-performance liquid chromatography (HPLC) analysis method used in this study. A

404

neighbor-joining phylogenetic tree based on the 16S rRNA gene sequences of A. faecalis CGMCC

405

17553 and closely related species is shown in Figure S1. Alignments of nitrilase sequences with

406

template sequences are provided in Figures S2, S3, S4, S5, and S6. Alignments of A. faecalis

20 / 43


Page 20 of 43

Page 21 of 43


407

CGMCC 17553 nitrilase active sites against selected nitrilase sequences are shown in Figures S7,

408

S8, S9, S10, and S11.

409

Funding

410

This research was financed by the National Science Foundation of China (grant no. 31570104)

411

and the Program for Jiangsu Excellent Scientific and Technological Innovation Team

412

(17CXTD00014).

413

Notes

414

The authors declare no competing financial interest.

21 / 43



1

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110

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1

Table 1. Primers used for the amplification of nitrilase genes from A. faecalis

2

CGMCC 17553. nitA-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGATGCTGGAATTGC GTCAG

nitA-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTACACTTCCTTGTC GTCCAGAAC

nitB-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGCAGACAAGAAAA ATCGTCC

nitB-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAGGACGGTTCCT GCACC

nitC-F

ACAGCAAATGGGTCGCGGATCCGAATTCGTGGTCATGAAAGTTG CATTGG

nitC-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAACCCAGTTCGG GCG

nitD-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGAGCAAAGTTGCTG TTATACAAG

nitD-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTTATTCGCCCGAGT CTTCG

nitE-F

ACAGCAAATGGGTCGCGGATCCGAATTCATGTCTACGACACGTG TTGCC

nitE-R

ATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCAAGAAGAAATAG GATACAGATCAC

28 / 43


Page 28 of 43

Page 29 of 43


1

Table 2. Degradation of flonicamid (initial concentration was 976.0 μmol/L) by

2

recombinant E. coli Rosetta (DE3) strains expressing nitrilase genes from A. faecalis CGMCC 17553.

3

Concentration (μmol/L) TFNG nitA

4 5 6

TFNG-AM

556.0±12.4 285.6±1.9

Reduced flonicamid 891.5±17.8a

nitB

ND

ND

12.9±10.1c

nitC

ND

ND

22.9±10.8c

nitD

ND

92.2±1.3

82.8±7.2b

nitE

ND

ND

21.9±9.7c

control

ND

ND

21.0±9.4c

Data represent the mean values of triplicate samples. Superscript letters indicate that the mean values (± SD) within that column are significantly different at p ≤ 0.05 based on analysis by Duncan’s test.

29 / 43



Page 30 of 43

1

Figure legends

2

Figure

3

chromatography-mass spectrometry (LC-MS) analysis of metabolites produced during

4

the degradation of flonicamid by A. faecalis CGMCC 17553. (A) HPLC analysis of the

5

transformation of flonicamid by A. faecalis CGMCC 17553. (B) Positive and (C) negative

6

ion mode mass spectrometry analysis of degradation product 1 (P1). (D) Positive and (E)

7

negative ion mode spectrometry analysis of degradation product 2 (P2).

8

Figure 2. Metabolic pathways of flonicamid and molecular structures used for substrate

9

specificity testing. (A) Various pathways for the metabolism of flonicamid. (B) Structure

1.

High-performance

liquid

chromatography

(HPLC)

and

liquid

10

of molecular used for substrate specificity testing.

11

Figure 3. Time course analysis of flonicamid degradation and kinetic parameters of the

12

reactions catalyzed by NitA and NitD. (A) Time course of flonicamid degradation by

13

resting A. faecalis CGMCC 17553 cells. (B) Degradation of flonicamid in soil by

14

faecalis CGMCC 17553. (C) Degradation of flonicamid by E. coli Rosetta (DE3)

15

expressing nitA from A. faecalis CGMCC 17553. (D) Degradation of flonicamid by E.

16

coli Rosetta (DE3) expressing nitD from A. faecalis CGMCC 17553.

17

Figure 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

18

analysis of NitA, NitB, NitC, NitD, and NitE overexpressed in E. coli Rosetta (DE3)

19

along with purified NitA and NitD. (A) SDS-PAGE analysis of NitA, NitB, NitC, NitD,

20

and NitE overexpressed in E. coli Rosetta (DE3). Lanes 1, 3, 5, 7, 9, and 11, total protein

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A.

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extracts from E. coli Rosetta (DE3) strains containing pET28a (control), pET28a-nitC,

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pET28a-nitB, pET28a-nitA, pET28a-nitD, and pET28a-nitE, respectively. Lanes 2, 4, 6,

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8, 10, and 12, soluble protein fractions from E. coli Rosetta (DE3) strains containing

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pET28a (control), pET28a-nitC, pET28a-nitB, pET28a-nitA, pET28a-nitD, and pET28a-

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nitE, respectively. Lane M, standard protein markers (116.0, 66.2, 45.0, and 35.0 kDa).

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(B) SDS-PAGE analysis of NitA expression in recombinant E. coli Rosetta (DE3) and

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purified NitA. Lane 1, soluble protein fraction from E. coli Rosetta (DE3) containing

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pET28a-nitA; lane 2, purified NitA with an N-terminal His-tag; lane M, standard protein

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markers (116.0, 66.2, 45.0, and 35.0 kDa). (C) SDS-PAGE analysis of NitD expression

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in recombinant E. coli Rosetta (DE3) and purified NitD, Lane 1, soluble protein fraction

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from E. coli Rosetta (DE3) containing pET28a-nitD; lane 2, purified NitD with an N-

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terminal His-tag; lane M, standard protein markers (116.0, 66.2, 45.0, and 35.0 kDa) .

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Figure 5. Enzymatic characterization of NitA. (A) Effects of pH on NitA activity. (B)

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Effects of pH on NitA stability. (C) Effects of temperature on NitA activity. (D) Effects

35

of temperature on NitA stability. (E) Effects of metalo ions on NitA activity. (F) Effects

36

of organic solvents on NitA activity. Superscript letters indicate that mean values (± SD)

37

within a column are significantly different at p ≤ 0.05 according to analysis by Duncan’s

38

test.

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Figure 6. Kinetic parameters of flonicamid degradation reactions catalyzed by NitA

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and NitD. (A) Kinetic parameters of TFNG formation by NitA. (B) Kinetic parameters of

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TFNG-AM formation by NitA. (C) Kinetic parameters of TFNG-AM formation by NitD. 31 / 43



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Figure 7. Enzymatic characterization of NitD. (A) Effects of pH on NitD activity. (B)

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Effects of pH on NitD stability. (C) Effects of temperature on NitD activity. (D) Effects

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of temperature on NitD stability. (E) Effects of metalo ions on NitD activity. (F) Effects

45

of organic solvents on NitD activity. Superscript letters indicate that mean values (± SD)

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within a column are significantly different at p ≤ 0.05 according to analysis by Duncan’s

47

test.

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Figure 8. Homology models of nitrilases from A. faecalis CGMCC 17553. (A) Three-

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dimensional homology model of NitA. (B) Three-dimensional homology model of NitB.

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(C) Three-dimensional homology model of NitC. (D) Three-dimensional homology

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model of NitD. (E) Three-dimensional homology model of NitE.

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Figure 9. Identification of actives sites within nitrilases from A. faecalis CGMCC

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17553. (A–E) Active sites in NitA, NitB, NitC, NitD, and NitE, respectively. Amino acid

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residues marked in green constitute the putative catalytic triad of each nitrilase. Amino

55

acid residues marked in yellow make up the active site of each nitrilase.

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TOC GRAPHICS

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Biodegradation of the insecticide flonicamid by ... - ACS Publications

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