Characterization of the propham biodegradation pathway in Starkeya

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Agricultural and Environmental Chemistry

Characterization of the propham biodegradation pathway in Starkeya sp. strain YW6 and cloning of a novel amidase gene mmH lina sun, Xinhua Gao, Wei Chen, Kaihua Huang, Naling Bai, Weiguang Lyu, and Hongming Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06928 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

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Journal of Agricultural and Food Chemistry

1

Characterization of the propham biodegradation pathway in Starkeya sp.

2

strain YW6 and cloning of a novel amidase gene mmH

3

Lina Sun1, 3, Xinhua Gao1, 6, Wei Chen1, 4, Kaihua Huang1, 6, Naling Bai1, 5, Weiguang

4

Lyu1, 3, 5 *, Hongming Liu2*

5

1

6

Agricultural Sciences, Shanghai, 201403, P.R. China

7

2

8

241000, P.R. China

9

3

Eco-Environmental Protection Research Institute, Shanghai Academy of

Institute of Molecular Biology and Biotechnology, Anhui Normal University, Wuhu,

Shanghai Engineering Research Center of Low-carbon Agriculture (SERCLA),

10

Shanghai, 201403, P.R. China

11

4

12

China

13

5

14

of Ministry of Agriculture, Shanghai, 201403, P.R. China

15

6

16

China

Shanghai Key Laboratory of Horticultural Technology, Shanghai, 201403, P.R.

Shanghai Agricultural Environment and Farmland Conservation Experiment Station

Environmental Protection Monitoring Station of Shanghai, Shanghai, 201403, P.R.

17 18 19 20 21 22

*Correspondence:

23

Weiguang Lyu

24

E-mail: [email protected]

25

Hongming Liu

26

E-mail: [email protected]

ACS Paragon Plus Environment


27

ABSTRACT

28

We previously isolated a monocrotophos-degrading strain Starkeya sp. YW6, that

29

could also degrade propham. Here, we show that strain YW6 metabolizes propham

30

via a pathway in which propham is initially hydrolyzed to aniline and then converted

31

to catechol, which is then oxidized via ortho-cleavage. The novel amidase gene mmH

32

was cloned from strain YW6 and expressed in E. coli BL21(DE3). MmH, which

33

exhibits aryl acylamidase activity, was purified for enzymatic analysis. Bioinformatic

34

analysis confirmed that MmH belongs to amidase signature (AS) enzyme family and

35

shares 26-50% identity with several AS family members. MmH (molecular mass of

36

53 kDa) was most active at 40°C and pH 8.0 and showed high activity toward

37

propham, with

38

characteristics make MmH suitable for novel amide biosynthesis and environmental

39

remediation.

40

KEYWORDS

41

MmH, Amidase, Propham, Gene cloning

Kcat and Km values of 33.4 s-1 and 16.9 μM, respectively. These

42 43 44 45 46 47 48 49 50 51


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52 53

INTRODUCTION Carbamate

pesticides,

which

are

esters

of

carbamic

acid,

inhibit

54

acetylcholinesterase (AChE) activity and thereby exert toxic effects on vertebrates

55

and invertebrates1-3. Carbamates are widely used as herbicides, insecticides,

56

nematicides and fungicides in agriculture4-5 . However, carbamate pesticides are

57

persistent in soils and natural waters, which leads to environmental contamination6-8.

58

Propham is a pre- and postemergence carbamate herbicide that is used to control

59

weeds in croplands of clover, alfalfa, sugar beets, flax, lettuces, spinach and peas9-10.

60

This herbicide inhibits cell division and the sprouting of meristematic tissue11-12.

61

Additionally, propham can be transformed to more harmful metabolites such as

62

aniline12-13. Propham has been classified as a group 3 carcinogenic compound (i.e.,

63

not classifiable based on its carcinogenicity to humans) by IARC Monographs14.

64

Therefore, the metabolic processes of this compound have attracted considerable

65

attention.

66

Microbes are important in the degradation of agrochemical pollutants in the

67

environment15-17. Several microorganisms that are able to hydrolyze propham have

68

been identified from different environments18-20. The fungus Beauveria sulfurescens

69

performs a specific parahydroxylation of the aromatic ring of propham21. The

70

blue-green alga Anacystis nidulans can convert this herbicide to anilines22.

71

Arthrobacter and Achromobacter species from soil are capable of mineralizing

72

propham via aniline23. Many strains that are capable of metabolizing aniline have also

73

been characterized24-27. However, to date, there has been no report of a strain that can

74

mineralize propham. Furthermore, the ligninolytic enzymes that transform propham to

75

aniline have been studied28, although a propham hydrolase gene has not been

76

identified.



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We previously isolated an efficient monocrotophos (MCP)-degrading strain,

78

Starkeya sp. strain YW629, which could also degrade propham, propanil,

79

2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA), 2-chloro-N-(2,6-diethyl

80

phenyl) acetamide (CDEPA) and chlorpropham. In this study, the degradation of

81

propham by strain YW6 was studied. A novel amidase gene, mmH, which encodes a

82

propham hydrolase responsible for the conversion of propham to aniline, was cloned

83

and characterized from strain YW6.

84

MATERIALS AND METHODS

85

Chemicals. Propham (99% purity) and propanil (98% purity)

86

Sigma-Aldrich Chemical Co. (Shanghai, China). CMEPA (98% purity) and CDEPA

87

(97% purity) were supplied by Syntechem Co., Ltd. (Jiangsu, China). Chlorpropham

88

(98% purity) was supplied by J&K Scientific Ltd. (Shanghai, China). All other

89

chemicals were of analytical grade.

90

Strains and plasmids. The strains and plasmids used in this study are listed in Table

91

1. Strain YW6 was previously isolated.

92

Culture conditions. All Escherichia coli strains were cultured in LB medium (Difco)

93

with appropriate antibiotics at 37°C. Strain YW6 was cultured at 30°C. Luria-Bertani

94

(LB) medium contained (in g l-1): 10.0 tryptone, 5.0 NaCl and 5.0 yeast extract (pH

95

7.0). Mineral salt medium (MSM) contained (in g l-1): 1.0 NH4NO3, 1.0 NaCl, 0.5

96

KH2PO4, 1.5 K2HPO4, 0.2 MgSO4·7H2O (pH 7.0) and 15.0 agar (for solid media).

97

Degradation experiments. To analyze the biodegradation of amide compounds,

98

strain YW6 was inoculated in LB medium and the cells were grown for 24 h. After

99

harvesting by centrifugation (4,000 rpm, 10 min, 4°C), we washed the cell pellet three

100

times with fresh MSM and suspended it in MSM (adjusting the cell density to

101

OD600nm = 1.0). Next the cell suspension was transferred to a 250-ml Erlenmeyer


were supplied by

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102

flask, which contained 50 ml of MSM and supplement with 50 mg l-1 of an amide

103

compound (propham, propanil, CMEPA, CDEPA, or chlorpropham). On a rotary

104

shaker (150 rpm) the cultures were incubated at 30°C. At 16-h intervals,

105

high-performance liquid chromatography (HPLC) was performed to analyze the

106

concentrations of pesticides and GC/MS was performed to identify their metabolites.

107

Control cultures inocubated under the same conditions without inoculation or

108

substrate addition were used as contrlis, and each treatment was carried out in

109

triplicate.

110

Cloning of the propham-hydrolyzing amidase gene (mmH). Genomic DNA was

111

extracted using the Bacterial Genomic DNA Fast Extraction Kit (DongSheng Biotech).

112

A genomic library of strain YW6 was constructed and plated on LB plates

113

supplemented with 500 µM 4-acetamidophenol and 100 mg l-1 ampicillin as described

114

by Yun et al30. Incubating for 12 h at 37°C, the plates were stored for 48 h at 16°C.

115

The colonies showing the target amidase-catalyzed hydrolysis of 4-acetamidophenol

116

to p-aminophenol31, were easily observed, since p-aminophenol is oxidized by

117

atmospheric oxygen to a purple-red product. Colonies with brown circles were

118

selected, and HPLC was performed to further assess their ability to degrade propham.

119

Insertion fragments from the positive clones were analyzed by the Beijing Genomics

120

Institute (Beijing, China), and the related nucleotide and protein sequences were

121

identified by BLASTN and BLASTP searches (www.ncbi.nlm.nih.gov/Blast).

122

OMIGA 2.0 software (Oxford Molecular Ltd.) was used to perform similarity

123

analyses of the protein sequences. The secondary structure of the protein was

124

predicted by JPred modeling server32. MEGA version 5.0 was used to perform

125

phylogenetic analyses of mmH with closely related arylamidases 33, and the topology

126

of the constructed tree was evaluated through a bootstrap analysis with 1200



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127

resamplings.

128

Expression and purification of MmH. We amplified the mmH gene from strain

129

YW6 genomic DNA and expressed it in E. coli with the pET29a expression system

130

(Novagen). The primer pairs used for amplification of mmH were mmH-F

131

(5'-GGGTTTCATATGGAAAGATCAGACTTGGACTATGC-3',

132

mmH-R

133

which included the Nde I and Xho I sites (underlined). Nde I and Xho I were used to

134

digest the amplified PCR product, which was subsequently gel purified and ligated

135

into pET29a. The generated recombinant plasmid pET-mmH was transformed into E.

136

coli BL21 (DE3). Subsequently, the resulting transformants were cultured in

137

autoinducing medium ZYM-5052 supplemented with 100 mg l-1 kanamycin at 28°C

138

for 16 h. We harvested, washed, suspendedand the cells in Tris-HCl buffer (20 mM,

139

pH 8.0), then disrupted them by sonication (Ultrasonic processor, model XL-2010).

140

The supernatant was applied to His-Bind resin (Novagen), the nontarget proteins were

141

eluted with 25 mM imidazole, and the target protein was eluted with 150 mM

142

imidazole. The enzyme was dialyzed against a solution containing Tris-HCl (20 mM,

143

pH 8.0) and 50 mM NaCl for 24 h, and then was concentrated using an Amicon

144

ultrafiltration.

145

(SDS-PAGE) was then used to analyze the purified enzyme.

146

Enzyme assay. The enzymatic activity assays were executed in 50 mM sodium

147

phosphate buffer (pH 8.0) containing substrate (200 μM) for 10 min at 40°C. The

148

reactions were initiated by adding purified MmH (50 μg ml-1) to 0.4 μg ml-1. One unit

149

of enzyme activity was defined as the amount of enzyme required to catalyze the

150

formation or hydrolysis of 1 μmol of product or substrate per min. For the kinetic

151

studies, substrates were diluted into six different concentrations. Data were fitted to

forward)

(5'-CCGCTCGAGCTGACTGCCAAGTGGACGGACCGC-3',

Sodium

dodecyl

sulfate-polyacrylamide


gel

and

reverse),

electrophoresis

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152

the Michaelis-Menten equation by OriginPro 8 software (OriginLab, Northampton,

153

MA). Kinetic parameters were obtained by nonlinear regression analysis via the

154

Michaelis-Menten equation.

155

The activities of aniline dioxygenase, catechol 2,3-dioxygenase and catechol

156

1,2-dioxygenase were tested as described by Liu et al.25. To assess the activity of

157

aniline dioxygenase, centrifugation was used to collect the cells at 13,700 × g, and

158

phosphate buffer (20 mM, pH 7.0) was used to wash them three times, then

159

resuspended. One unit of aniline dioxygenase activity was defined as the weight of

160

wet cells that consumed 1 μmol of oxygen per min. The cells were disrupted by

161

sonication for 5 min, and the lysate was subsequently centrifuged for 20 min at 13,700

162

× g and 4°C. The supernatant was used for catechol 1,2-dioxygenase and catechol

163

2,3-dioxygenase assays.

164

The optimal reaction pH was determined in three differernt pH ranges of buffer as

165

follows: sodium acetate buffer (pH 4.0-6.5), potassium phosphate buffer (pH 6.0-8.0)

166

and Tris-HCl buffer (pH 7.5-11.0). To determine the pH stability of MmH, it was

167

incubated for 1 h at 40°C in buffers with different pH values (4.0 to 11.0), after which

168

the enzyme residual activity was tested using the method described above. The

169

optimum reaction temperature was tested within a temperature range of 10-70°C. To

170

determine the thermal stability of MmH, MmH was preincubated for 1 h at different

171

temperatures in a water bath, after which the residual activity of the enzyme was

172

tested. All of the assays were performed in triplicate.

173

The effects of potential inhibitors or activators on MmH activity were tested by

174

adding various metal salts (Mg2+, Ca2+, Cu2+, Mn2+, Ni2+, Zn2+, Co2+ and Hg2+) and

175

chemical agents (Tween-80, EDTA, Triton X-100, SDS, iodoacetamide and

176

mercaptoethanol) at 1 mM to the reaction mixtures, which had been preincubated at



177

40°C for 10 min. Control reactions without enzyme or additive were performed in

178

parallel. The activity of MmH was tested as described above.

179

Chemical analysis. Ethyl acetate was used to extract the culture medium and

180

enzymatic hydrolysate. Anhydrous sodium sulfate was used to dry the extract, and

181

ethyl acetate was evaporated under decompression. Methanol was used to dissolved

182

the residues in 1 ml. HPLC was used to test the concentrations of chemical

183

compounds by a Zorbax C18 ODS Spherex column (250 mm × 4.6 mm, Agilent).

184

Methanol-water (80:20, v/v) was used as the mobile phase, the flow velocity was 1.0

185

ml min-1 and the detection wavelength was 230 nm. A GC/MS system (Finnigan,

186

ThermoFisher, San Jose, CA, USA) equipped with an MS detector was used to test

187

the metabolites in electron ionization mode (70 eV). The Finnigan capillary column

188

(15 m × 0.5 mm × 0.25 μm) temperature was programmed as follows: 80°C for 2 min,

189

increased to 280°C at 25°C min-1, and then held for 5 min. Helium was used as the

190

carrier gas with an invariable flow rate of 1.0 ml min-1. The external standard

191

calibration method was used to quantify the amount of substrate and product.

192

GenBank accession number. The GenBank accession number of the mmH gene is

193

KT894939.

194

RESULTS AND DISCUSSION

195

Degradation of propham by Starkeya sp. strain YW6. We previously isolated

196

strain YW6 from paddy soil in Hunan Province, PR China29, and showed strain YW6

197

was also able to degrade propham. The cell suspension of strain YW6 degraded 100%

198

of the propham supplied within 24 h. (Figure 1A). As shown in Figure 2C, one

199

product with a retention time (RT) of 5.601 min appeared during propham

200

degradation. This metabolite had the same RT as authentic aniline (Figure 2B). As

201

shown in Figure 3, one metabolite was detected during the degradation of propham.


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202

Every compound was identified by its mass spectrum using the NIST library program.

203

The mass spectral data for compound Ⅰ (RT = 7.53 min) demonstrated that this

204

compound was propham. Compound Ⅱ (RT = 4.11 min) exhibited a molecular ion

205

peak at m/z 93.10 [M]+ , which corresponds to the cleavage of the amide bond of

206

propham. Thus, this compound was proposed to be aniline. Aniline accumulation was

207

accumulated during the first 12 h (Figure 1A), and after this time point, the

208

concentration of aniline began to decrease, which suggested that strain YW6 can

209

degrade propham to aniline. To identify the mechanism by which strain YW6

210

degrades aniline, the aniline dioxygenase, catechol 1,2-dioxygenase and catechol

211

2,3-dioxygenase activities in cellular lysates acquired from strain YW6 cultured in the

212

presence of aniline or glucose were determined (Table 2). When cultured with 50 mg

213

l-1 aniline, strain YW6 exhibited high levels of aniline dioxygenase and catechol

214

1,2-dioxygenase activity compared with that observed without glucose, whereas

215

catechol 2,3-dioxygenase activity was not detected. These results indicate that aniline

216

dioxygenase and catechol 1,2-dioxygenase are key enzymes for the biodegradation of

217

propham in strain YW6. Based on the results of enzymatic studies and metabolite

218

identification, the following complete metabolic pathway for propham can be

219

proposed. In strain YW6, the propham was hydrolyzed first by the amidase MmH

220

yielding aniline. Aniline induced aniline dioxygenase and catechol 1,2-dioxygenase

221

activities, suggesting that aniline was further degraded to catechol, which was

222

subsequently oxidized via an ortho-cleavage pathway. The propham degradation

223

pathway used by strainYW6 identified based on the abovementioned results is shown

224

in Figure 4.

225

Starkeya sp. strain YW6 exhibits degradation activity toward propham,

226

chlorpropham, propanil and monocrotophos, which could be useful for the



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227

bioremediation of environments polluted with multiple pesticides. In addition, the

228

chloroacetamide herbicides alachlor, acetochlor, pretilachlor, butachlor and

229

metolachlor can be transformed to CMEPA and CDEPA by N-demethylase in certain

230

strains, such as Rhodococcus sp. B1 and T3-117, 34. However, strains T3-1 and B1

231

cannot degrade the products CMEPA and CDEPA. Strain YW6 could also degrade

232

CMEPA and CDEPA, which could facilitate the degradation of chloroacetamide

233

herbicides in the environment.

234

Amidase gene cloning and sequence analysis. From approximately 8000

235

transformants, we obtained one brown colony that was demonstrated to hydrolyze

236

propham. The 4775-bp fragment in the positive clone, was observed to harbor 28

237

open reading frames (ORFs), and one of which shared high identity with several

238

hypothetical or putative amidase genes via BLASTP. We subsequently subcloned the

239

ORF into pMD18-T, then transformed it into E. coli DH5α, and found that the

240

transformant hydrolyzed 4-acetaminophenol and propham via HPLC. The ORF

241

encoding the propham hydrolase was named mmH.

242

The sequence analysis results suggested that the mmH ORF consisted of 1479 bp

243

and encoded a 492-amino-acid protein. The G+C content was 60%. The start codon of

244

the ORF was TTG, and the stop codon was TAG. The molecular weight of mmH was

245

52,467 Da as determined by proteomic predictions (ExPASy). The BLASTP program

246

was used to search the predicted protein sequence from the Protein Data Bank (PDB;

247

http://blast.ncbi.nlm.nih.gov). MmH shows a high level of similarity with amidases

248

and 50% protein sequence identity with p-nitroacetanilide hydrolase AAA, which is

249

the highest identity. MmH is a member of the amidase family, exhibiting a highly

250

conserved

251

(GGSSGGTSAAVADGLLPIGDGTDGGGS, sites 157 to 183). Furthermore, the

serine-

and

glycine-rich


sequence

Page 11 of 26


252

sequence analysis revealed that MmH harbors the sequence Ser183-Ser159-Lys82,

253

which is a highly conserved catalytic triad that is present in amidase family members.

254

Thus, MmH belongs to the amidase signature (AS) enzyme family. Phylogenetic

255

analysis of MmH showed that it is related to but different from propanil hydrolase

256

PamH (30% identity), p-nitroacetanilide hydrolase AAA (50% identity), and other

257

hydrolases (< 30% identity) (Figure 5).

258

Expression, purification and characterization of MmH. E. coli BL21 (DE3) was

259

used to express MmH, and Ni-nitrilotriacetic acid affinity chromatography was used

260

for purification. MmH-His6 was purified, and appeared as a single band in

261

SDS-PAGE (Figure 6). The denatured enzyme had a molecular weight of about 53

262

kDa, which was the same molecular weight as the calculated amino acid sequence

263

(52,467 Da). Furthermore, MmH catalyzed the hydrolysis of propham (Figure 2D).

264

The optimal pH for MmH was determined to be 8.0, and from pH 7.0 to 10.0 MmH

265

was highly active. MmH was stable between pH 6.0 and 10.0 (Figure S1). Afer a 1 h

266

preincubation at these pH value, MMH retained more than 65% of its original

267

activity. The optimum temperature for MmH activity was 40°C (Figure S2). After a 1

268

h preincubation at 50°C, MmH retained more than 60% of its original activity,

269

indicating that MmH was fairly stable at temperatures up to 50°C (Figure S2), and at

270

70°C, it was completely inactivated.

271

Hg2+ (1 mM) severely inhibited MmH activity, whereas the addition of Mg2+ and

272

Mn2+ increased its acivity by 1.18- and 1.13-fold, respectively. Other tested metal ions

273

showed 10-40% inhibition of amidase activity. Iodoacetamide, mercaptoethanol,

274

Triton X-100, Tween-80 and the surfactants SDS induced 30-50% inhibition of MmH

275

activity. However, the chelating agent EDTA (10 mM) inhibited the activity of MmH

276

by only 4.2%, suggesting that a divalent cation is not required for MmH activity



277

(Table 3).

278

Substrate spectrum. Various amide compounds were used to examine the substrate

279

specificity of MmH (Table 3). MmH could hydrolyze CMEPA, which is a crucial

280

metabolic intermediate in the biodegradation of acetochlor35. MmH could catalyze the

281

hydrolysis of chlorpropham to generate the corresponding 3-chloroaniline. However,

282

this enzyme could not hydrolyze butachlor, acetochlor or other structurally analogous

283

compounds. The results showed that propham was an appropriate substrate for MmH.

284

Under optimal conditions, the Kcat and Km values obtained for propham were 33.4 s-1

285

and 16.9 μM, respectively, and the catalytic efficiency (Kcat/Km) was 1.97 μM-1 s-1.

286

GC/MS was used to analyze the metabolites of CMEPA, propanil, chlorpropham and

287

CDEPA (Figure S3 to S6).

288

During the microbial degradation of amide pesticides, amide bond hydrolysis is a

289

vital reaction. To date, several amidases important in the degradation of amide

290

pesticides have been cloned and characterized36-37. These enzymes contain the GGSS

291

motif in the primary structure and a unique, highly conserved Ser-Ser-Lys catalytic

292

triad that is characteristic of amidase38-39. A novel amidase gene, mmH, was identified

293

in Starkeya sp. strain YW6, and the recombinant enzyme MmH exhibited amidase

294

activity, thus enriching hydrolase gene diversification. MmH has a broad substrate

295

spectrum, which coupled with its tolerance to a range of pH values, makes strain

296

YW6 useful for environmental remediation and novel amide biosynthesis.

297

Supporting Information Figure S1 Effect of pH on the enzyme activity and stability

298

of MmH. Figure S2 Effect of temperature on the enzyme activity and stability of

299

MmH. Figure S3 GC/MS analysis of CMEPA hydrolysis by MmH. Figure S4 GC/MS

300

analysis of propanil hydrolysis by MmH. Figure S5 GC/MS analysis of chlorpropham

301

hydrolysis by MmH. Figure S6 GC/MS analysis of CDEPA hydrolysis by MmH.


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302

Funding Sources

303

This work was supported by the National Natural Science Foundation of China (No.

304

31700097); Shanghai Sailing Program (No. 17YF1416900); Shanghai Sailing

305

Program (No. 18YF1420900); Domestic Cooperation Program of Shanghai Science

306

and Technology Commission (No. 18295810500); Key Agricultural Technology

307

Program of Shanghai Science and Technology Commission (No. 16391901500);

308

Shanghai

309

(No.T20180414); SAAS Program for Excellent Research Team (nongkechuang

310

2017A-03) and State Key Laboratory Breeding Base for Zhejiang Sustainable Pest

311

and Disease Control (No. 2010DS700124-KF1813).

312

Notes

313

The authors declare no competing financial interests.

Agriculture

Applied

Technology

Development

314 315 316 317 318 319 320 321 322 323 324 325 326 327


Program,

China


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Page 18 of 26

Figure Captions Figure 1. (A) Dynamics of the degradation of propham in MSM by strain YW6: (●) degradation of propham; (▲) accumulation of aniline; (■) growth of strain YW6. (B) Dynamics of the degradation of catechol in MSM by strain YW6: (□) degradation of catechol; (△) growth of strain YW6.

Figure 2. Identification of propham and its putative degradation intermediates by comparing the HPLC retention times with those of standard chemicals. (A) Propham standard. (B) Aniline standard. (C) Propham degraded by strain YW6. (D) Propham hydrolyzed by MmH.

Figure 3. GC/MS analysis of propham transformation by strain YW6: (A) GC profiles for propham and its metabolites. The retention times of the compounds were 7.53 and 4.11 (B-C) Characteristic ions of compounds Ⅰ and Ⅱ obtained by GC/MS; the compounds were identified as propham and aniline, respectively.

Figure 4. Proposed metabolic pathway of propham in strain YW6.

Figure 5. Phylogenetic tree of MmH and some related arylamidases constructed by the neighbor-joining method. AAA (Pseudomonas sp., p-nitroacetanilide), AmpA ( Paracoccus sp. strain FLN-7, diflubenzuron), LibA (Variovorax sp. strain SRS16, linuron), PamH (Paracoccus sp. strain M-1, propanil), OctHD (Rhodococcus sp. strain

Oct1,

o-nitroacetanilide),

NylA

(Arthrobacter


sp.

strain

KI72,

Page 19 of 26


6-aminohexanoate

cyclic

dimer

NylA/laurolactam),

ChnC

(Arthrobacter

nitroguajacolicus Rü61a, N-acetylanthranilate), DamH (Delftia sp. T3-6, CMEPA), GatA, and CmeH (Sphingobium quisquiliarum DC-2, CMEPA). Numbers at nodes are percentage bootstrap values based on 1200 resampled datasets. The scale bars indicates 0.2 substitution per nucleotide position.

Figure 6. SDS-PAGE of purified MmH-His6. Lane M, molecular mass standards (molecular masses are indicated on the left in kDa); lane 1, purified MmH-His6.



Page 20 of 26

Table 1．Strains and plasmids used in this study. Strains and plasmids

Characteristic(s)

Source or reference

Starkeya sp. YW6

Wild type

E. coli DH5α

Host strain for cloning vectors

Lab stock

E. coli BL21 (DE3)

Expression host

Lab stock

pUC 118 BamHI/BAP

BamHI-digested DNA fragment

TaKaRa

pUC 118-YW6

cloning vectors pUC 118 derivative carrying the

This study

29

Sau3AI-digested DNA fragment pT-mmH

pUC 18-Tcontaining derivativemmH carrying mmH

This study

pET-29a(+)

Expression vector

Novagen

pET-mmH

pET-29a(+) derivative carrying mmH

This study

Table 2. Specific activities of enzymes in the cells or cell-free extracts of strain YW6 cultured in MSM with different carbon sources. Growth substrates

Aniline dioxygenase (U/mg of wet cell)

Catechol-1,2 dioxygenase (U/mg of protein)

Catechol-2,3-dioxygenase (U/mg of protein)

Aniline Glucose

0.68±0.05 -

1.35±0.1 -

-

-, no activity was detected.


Page 21 of 26


Table 3． Effect of metal ions on the enzyme activity of MmH Reagent

Concentration

Relative activity (%)

No addition

0 mM

100.0

Ca2+

1 mM

89.7±1.1

Co2+

1 mM

85.5±0.7

Cu2+

1 mM

65.9±0.5

Hg2+

1 mM

21.9±0.3

Zn2+

1 mM

60.4±0.9

Mg2+

1 mM

118.1±1.7

Mn2+

1 mM

113.2±0.4

Ni2+

1 mM

70.4±0.9

Iodoacetamide

1 mM

58.2±0.7

EDTA

10 mM

95.8±1.5

Mercaptoethanol

10 mM

65.9±1.8

SDS

2 mg/ml

69.7±0.3

Triton X-100

20 mg/ml

53.5±0.6

Tween 80

20 mg/ml

63.3±1.4

Table 4．Substrate specificity and apparent kinetic constants of MmH Catalytic efficiency Substrate

Kcat (s-1)

Km (μM)

(Kcat/Km [μM-1 · s-1])

CMEPA

31.8±2.2

19.7±1.2

1.61

Propanil

32.7±1.5

30.2±2.1

1.08

Chlorpropham

33.7±1.9

41.6±1.8

0.81

CDEPA

33.1±1.2

20.5±1.7

1.61

Propham

33.4±1.4

16.9±1.1

1.97



Figure 1.

A

B


Page 22 of 26

Page 23 of 26


Figure 2. A

3.027

Intensity (AU)

B A A

5.602

C A A

3.021

5.601

D A A

3.026 5.587

Retention time (min)



Page 24 of 26

Figure 3. A Ⅰ Ⅱ

B

C

Ⅰ

Ⅱ

Figure 4.

MmH

aniline dioxygenase

catechol 1, 2-dioxygenase

H2O (CH3)2CHOCOOH

H2O+CO2


Page 25 of 26


Figure 5. AAA (ACP39716.2)

48 99

MmH (KT894939) GatA (BAH90190.1)

95

PamH (AEF33439) AmpA (JQ388838)

99

NylA (YP001965070) OctHD (BAI44731.1)

69

DamH (AEO20074.1)

99 99

LibA (AEO20074.1) ChnC (AAG10029.1) CmeH (KF294465)

99

0.2

Figure 6. kDa

M

1

116 66.2 45

MmH-His6

35

25 18.4 14.4



TOC graphic


Page 26 of 26

Characterization of the propham biodegradation pathway in Starkeya

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