A Rieske-Type Oxygenase of Pseudomonas sp. BIOMIG1 Converts

Subscriber access provided by SAN DIEGO STATE UNIV

Article

A Rieske-type Oxygenase of Pseudomonas sp. BIOMIG1 converts Benzalkonium Chlorides to Benzyldimethyl Amine Emine Ertekin, Konstantinos T. Konstantinidis, and Ulas Tezel Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03705 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Environmental Science & Technology

1

A Rieske-type Oxygenase of Pseudomonas sp. BIOMIG1

2

converts Benzalkonium Chlorides to Benzyldimethyl Amine

3 4

Emine Ertekin1, Konstantinos T. Konstantinidis3,4 and Ulas Tezel1,2,*

5 6

1

7

TURKEY

8

2

9

TURKEY

Institute of Environmental Sciences, Bogazici University, Bebek 34342 Istanbul,

Center for Life Sciences and Technologies, Bogazici University, Bebek 34342 Istanbul.

10

3

11

Atlanta GA, 30332-0512, USA

12

4

13

*Corresponding author. Phone: +90 212-359-4604; fax: +90 212-257-5033;

14

e-mail address: [email protected] (U. Tezel)

School of Civil and Environmental Engineering, Georgia Institute of Technology,

School of Biology, Georgia Institute of Technology, Atlanta GA, 30332-0512, USA

15 16

ABSTRACT

17

Recently, an array of eight genes involved in the biotransformation of benzalkonium

18

chlorides (BACs) – an active ingredient of many disinfectants - to benzyldimethyl amine

19

(BDMA) was identified in the genome of Pseudomonas sp. BIOMIG1, which is a

20

bacterium present in various environments and mineralizes BACs. In this study, we

21

showed that heterologous expression of an oxygenase gene (oxyBAC) present in this gene

22

array in E. coli resulted in formation of BDMA from BACs at a rate of 14 µM h-1.

23

oxyBAC is phylogenetically classified as a Rieske-type oxygenase (RO) and belongs to a 1 ACS Paragon Plus Environment


Page 2 of 28

24

group which catalyzes the cleavage of C-N+ bond between either methyl or alkyl ester

25

and a quaternary nitrogen (N) of natural QACs such as stachydrine, carnitine and

26

trimethylglycine. Insertion of two glycine into the Rieske domain and substitution of

27

tyrosine with leucine in the mononuclear iron center differentiate oxyBAC from other

28

ROs that cleaves C-N+, and presumably facilitate the cleavage of saturated alkyl chain

29

from quaternary N via N-dealkylation reaction. In addition, unlike other ROs, oxyBAC

30

did not require a specific reductase to function. Our results demonstrate that oxyBAC

31

represents a new member of RO associated with BAC degradation, and have implications

32

for controlling the fate of BACs in the environment.

33 34

INTRODUCTION

35

Quaternary ammonium compounds (QACs) are cationic surfactants that are used as

36

disinfectants in industrial, domestic and medical applications extensively since 1930s1 .

37

Benzalkonium chlorides (BACs) are the most commonly used group of QACs1. Due to

38

their unrestricted use since decades, BACs are continuously discharged into the

39

environment either directly or through wastewater treatment plants, which are not

40

designed to treat such pollutants like BACs. As a result, BACs are present in every

41

compartment of the environment at concentrations ranging from 0.05 mg L-1 in surface

42

waters 2 to 5 mg L-1 in hospital effluents 2, 3. Contamination of the environment with

43

BACs can cause severe ecological and public health problems such as eco-toxicity 1 and

44

dissemination of antimicrobial resistance4.

45 46

Despite their antimicrobial properties, some microorganisms in the environment can degrade BACs and convert them to non-toxic end products 5-7. BAC degradation

2 ACS Paragon Plus Environment

Page 3 of 28


47

commences with an initial attack to the alkyl chain, which cleaves the C-N bond. This N-

48

dealkylation reaction results in the formation of benzlydimethyl amine (BDMA) and an

49

alkanaldehyde as intermediates. As BDMA is approximately 500 times less toxic than

50

BAC8, this is the key step in BAC biotransformation that alleviates the environmental

51

impact of BACs.

52

Several microorganisms capable of catalyzing this reaction have been

53

characterized to date9-11. However, there is relatively limited information on the genetics

54

of QAC N-dealkylation reaction achieved by those microorganisms. To date, tetradecyl

55

trimethyl ammonium bromide monooxygenase (TTABMO) of Pseudomonas putida

56

ATCC 12633 12 and amine oxidase of a Pseudomonas nitroreducens B 13 which

57

converted QACs to tertiary amines have been characterized. TTABMO is a typical

58

flavoprotein that utilizes nicotinamide adenine dinucleotide phosphate (NADPH) and

59

flavin adenine dinucleotide (FAD) as cofactors, whereas the amine oxidase of

60

Pseudomonas nitroreducens B requires only FAD. Phylogenetically, amine oxidase is

61

more closely related to Pseudomonas sp. pseudooxynicotine amine oxidase14, which

62

catalyzes deamination of pseudooxynicotine, rather than TTABMO, although both of

63

them are QAC N-dealkylating ezymes. In addition, three genes encoding oxygenases that

64

metabolized glycine betaine (gbcA), stachydrine (stc2) and carnitine (cntA), which are

65

naturally occurring QACs, have been identified15, 16. Those enzymes are Rieske non-

66

heme iron dependent oxygenases that catalyze the N-dealkylation of QACs (Figure S1).

67

Among them, stc2 and gbcA removes methyl group from quaternary N (Figure S1A and

68

B) whereas cntA cleaves the C-N+ bond between an ester group and the quaternary N



Page 4 of 28

69

(Figure S1C). However none of them were shown to attack on C-N+ between a saturated

70

alkane and quaternary N that is present in synthetic QACs such as BACs.

71

Recently, we isolated a new BAC-degrader, Pseudomonas sp. BIOMIG1, and

72

demonstrated that an array of genes including multidrug transporters, a transcriptional

73

regulator, a sterol binding domain protein, an integrase and an oxygenase, all encoded on

74

the same piece of DNA, were essential for BAC degradation. Moreover, the same array

75

of genes was also highly abundant in BAC degrading microbial communities originating

76

from four different environments 17. The only catabolic gene present in this array encoded

77

a predicted oxygenase (oxyBAC), which was not similar to any other QAC degrading

78

enzymes previously identified. In this study, our objective was to elucidate the function

79

of the oxygenase gene identified in the BIOMIG1 by genetic manipulations using

80

heterologous expression of the gene in E.coli. Therefore our study identified a new gene

81

and the corresponding enzyme involved in the QAC degradation, which might be

82

widespread in the natural and engineered systems.

83 84

MATERIALS AND METHODS

85

Microorganisms. Pseudomonas sp. BIOMIG1BAC (GenBank Accession No:

86

MCRS00000000) which has been shown to mineralize BACs and BDMA and

87

Pseudomonas sp. BIOMIG1BDMA (MCRT01000000) which converts BACs to BDMA but

88

cannot utilize BDMA were isolated from a BAC degrading enrichment community

89

developed from sewage taken from a local wastewater treatment plant in Istanbul,

90

Turkey17. In addition, Pseudomonas sp. BIOMIG1BD (MCRU01000000) which utilizes

91

BDMA but not BACs has been generated from Pseudomonas sp. BIOMIG1BAC by serial


Page 5 of 28


92

culturing on a salt medium (SM) containing only BDMA as the carbon source 17.

93

Pseudomonas sp. BIOMIG1N (MCRV00000000) that degrades neither BACs nor BDMA

94

has been generated by culturing the Pseudomonas sp. BIOMIG1BDMA only in LB. E.coli

95

BL21(DE3) pLysS (Invitrogen, Thermo Fisher Scientific, USA) was used in heterologous

96

expression experiments. Summary of the strains and their characteristics are given in

97

Table 1.

98

Bioinformatic identification and classification of BAC oxygenase. In order to identify

99

the genes involved in the BAC conversion to BDMA, genomes of aforementioned four

100

BIOMIG1 phenotypes were compared as described elsewhere17. Briefly, the gene

101

sequences in the genomes were compared in a pairwise manner using “reciprocal best

102

match blast”. The set of genes that was present in the genome of the Pseudomonas sp.

103

BIOMIG1BAC and Pseudomonas sp. BIOMIG1BDMA but absent in the Pseudomonas sp.

104

BIOMIG1BD and Pseudomonas sp. BIOMIG1N was determined as the candidate genes

105

involved in BAC to BDMA transformation. An array composed of 8 genes was identified.

106

An oxygenase was the only catabolic gene in this array and selected as the candidate gene

107

responsible for BAC transformation to BDMA via N-dealkylation reaction. This gene

108

was mentioned as oxyBAC here after.

109

The amino acid sequence of oxyBAC (Text S1) was annotated using BLASTP in

110

the non-redundant protein sequence database of “National Center of Biotechnology

111

Information” (NCBI, http://www.ncbi.nlm.nih.gov/protein). For the phylogenetic

112

classification of oxyBAC, protein sequences of homolog oxygenases along with

113

previously identified QAC degrading enzymes were used. Briefly, protein sequences



114

were aligned using ClustalW implemented in Geneious® 7.1 (Amsterdam, Netherlands)

115

and Jukes-Cantor neighbor joining method was used for clustering.

Page 6 of 28

PCR primers for oxyBAC were designed to verify the presence of the gene in the

116 117

BIOMIG1 phenotypes as well as the in the E.coli host used in heterologous expression

118

experiments with Primer 3 module in Geneious® 7.1 (Amsterdam, Netherlands) using

119

the gene sequences identified by genome comparison. The primer sequences are provided

120

in Table 1. The amplification conditions were as follows; denaturation at 95°C for 10

121

seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 1 minute, for 30

122

cycles.

123

BAC biotransformation experiments with Pseudomonas sp. BIOMIG1 phenotypes.

124

Pseudomonas sp. BIOMIG1BAC and Pseudomonas sp. BIOMIG1BDMA were grown in LB

125

containing 50 mg L-1 BACs (Barquat 80, Lonza Inc., Switzerland). An overnight grown

126

culture of each phenotype was resuspended in SM8, 17 and spiked with 150 µM BAC

127

containing equimolar amounts of dodecyl benzyl dimethyl ammonium chloride

128

(C12BDMA-Cl, TCI Chemicals, Japan) and tetradecyl benzyl dimethyl ammonium

129

chloride (C14BDMA-Cl, TCI Chemicals, Japan). After all BAC was consumed, cultures

130

were diluted to OD600 of 0.02 AU (~106 cells mL-1) in SM and respiked with 300 µM

131

BACs. The cultures were incubated at room temperature on a rotary shaker at 130 rpm.

132

Samples were taken intermittently to monitor BAC concentration by HPLC using the

133

method described elsewhere17, and cell growth was measured by plate counting. On the

134

other hand, BIOMIG1BD and BIOMIG1N were grown in LB with 1000 mg L-1 BDMA or

135

only in LB, respectively. The overnight grown cultures were resuspended in SM to an


Page 7 of 28


136

OD600 of 0.2 AU, and spiked with 300 µM BAC. BAC concentration and cell density

137

were measured as described above.

138

Heterologous expression of genes in E. coli. The oxyBAC gene was chemically

139

synthesized by GenScript and inserted into pet29a+ vector (Merck Millipore, Germany),

140

between the NdeI and NcoI restriction sites, respectively (Figure S2). The E.coli

141

BL21(DE3) pLysS strain (Invitrogen, Thermo Fisher Scientific, USA) was transformed

142

with plasmid containing oxyBAC (E. colioxyBAC) (Table 1). For protein overexpression,

143

E.coli cells were grown in LB broth containing 30 µg mL-1 kanamycin (Sigma-Aldrich,

144

Germany) and 34 µg mL-1 chloramphenicol (Sigma-Aldrich, Germany) at 37°C. After the

145

turbidity reached at OD600 of 0.5, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)

146

(Sigma-Aldrich, Germany) was added to initiate induction, which was carried out at room

147

temperature for 24 hours. Expression of the 43 kDa oxyBAC protein was confirmed

148

using SDS-PAGE of crude protein extracts (Figure S3).

149

Following induction, E. colioxyBAC cells were harvested and inoculated into 20 mL

150

SM containing 20 µM of C12BDMA-Cl at a final O.D. of 2.0. At determined intervals,

151

C12BDMA-Cl utilization and BDMA production were monitored by HPLC as described

152

by Ertekin et al. (2016)17. A control assay was performed with E. coli cells transformed

153

with the pet29a+ plasmid not containing oxyBAC gene (E. coliN, Table 1). To calculate

154

biotransformation rates, experimental data was fitted to Michaelis-Menten growth model

155

using Berkeley-Madonna software employing Runge-Kutta 4 integration method, as

156

described by Ertekin et al. (2016)17.

157 158



Page 8 of 28

159

RESULTS AND DISCUSSION

160

Phylogenetic analysis and classification of oxyBAC. Analysis of the amino acid

161

sequence of oxyBAC confirmed that it encoded a conserved Rieske domain [2Fe-2S] and

162

an iron containing catalytic active site, and therefore this protein was identified as a

163

Rieske oxygenase (RO). Nam et al. (2001)18 classified ROs into four distinct groups

164

according to the amino acid sequence patterns of their oxygenase subunits. Those four

165

RO groups (Group I-IV, Figure 1) mainly catalyze cis and trans hydroxylation of a

166

benzene ring. However, it was recently reported that some of the newly discovered ROs

167

did not cluster with any of these groups, thus representing a novel group. The ROs that

168

belonged to the latter group include enzymes like stachydrine demethylase (stc2)15,

169

glycine demethylase (gbcA)19 and carnitine oxygenase (cntA)16, which could carry out

170

non-ring hydroxylating reactions. More specifically, the latter enzymes attack on C-N+

171

bond between a methyl or ester groups and quaternary N of natural QACs resulting in

172

removal of these groups from the N atom and formation of a tertiary amine (Figure S1A,

173

B and C) In order to dealineate the evolutionary relationship of oxyBAC with the

174

previously characterized ROs, a phylogenetic tree was constructed using the amino acid

175

sequences of its closest homologs along with two recently identified QAC degrading

176

enzymes (Figure 1). Our results demonstrated that oxyBAC belonged to a distinct clade

177

including stc2, gbcA and cntA which catalyze N-dealkylation of aforementioned natural

178

QACs. This group was further denoted as Group V ROs in this study. The previously

179

identified enzymes degrading QAC disinfectants such as the amine oxidase of

180

Pseudomonas nitroreducense B6 and tetradecyltrimethylammonium bromide


Page 9 of 28


181

monooxygenase of Pseudomonas putida ATCC 12633 (TTBMO) 12 are non-RO enzymes

182

and were more closely related to Group II ROs as opposed to Group V enzymes.

183

Furthermore, among ROs, Group V was more closely related to monooxygenases

184

such as the choline monooxygenase (cmo). On the other hand, cmo cannot catalyze a N-

185

dealkylation reaction but can oxidize the terminal C of the alcohol group attached to the

186

quaternary N of the choline (Figure S1D) 20. Within this group, oxyBAC had 26% amino

187

acid similarity to stc2 which was its closest biochemically characterized homolog. This

188

relatively low homology to previously characterized enzymes doing N-dealkylation of

189

short saturated and un-saturated alkyl groups suggests that oxyBAC is a novel enzyme,

190

potentially involved in the N-dealkylation of a long-chain saturated alkyl group from a

191

quaternary N (Figure S1E). The previously identified BAC dealkylating amine oxidase6

192

and TTABMO12 showed substantially lower homology to oxyBAC (13% and 17%,

193

respectively) compared to cntA, gbcA and stc2.

194

The most similar amino acid sequence (~97% amino acid similarity) to oxyBAC

195

was present in the draft genome of Novosphingobium sp. B7 (GenBank Accession

196

Number: WP_022676119.1). The gene encoding this protein was located on a 3 kbp long

197

contig. The sequence of this contig was almost identical to the gene cluster containing

198

oxyBAC (98% nucleotide similarity). However, neither Novosphingobium sp. B7, nor the

199

aforementioned protein has been previously tested for QAC degradation. The flanking

200

regions of oxyBAC in our Pseudomonas sp. BIOMIG1 strain consisted of two MFS

201

transporters, a tetR family type transcriptional regulator, a sterol binding domain protein,

202

a hypothetical protein, a Lyrs family transcriptional regulator and a phage integrase17.

203

This structure was also conserved in the genome of Novosphingobium sp. B7, where the



Page 10 of 28

204

homolog oxygenase was flanked with a tetR family transcriptional regulator, a sterol

205

binding protein and two hypothetical proteins. In the latter gene cluster, a reductase gene

206

next to oxyBAC was not identified which is not usual for ROs. The latter finding

207

indicated that oxyBAC could function without a specific reductase or recruit a non-

208

specific reductase for electron transfer.

209

Pseudomonas sp. BIOMIG1 with oxyBAC grows on BACs. We confirmed that

210

oxyBAC gene was present in the genomes of Pseudomonas sp. BIOMIG1BAC and

211

BIOMIG1BDMA, but absent in the genomes of Pseudomonas sp. BIOMIG1BD and

212

Pseudomonas sp. BIOMIG1N by amplifying a ~700 bp region of the gene by PCR using a

213

specific primer set (Figure 2A). Given that BIOMIG1BD and BIOMIG1N were generated

214

from BIOMIG1BAC and BIOMIG1BDMA, respectively by selective cultivation in BAC free

215

media, it can be assumed that oxyBAC was lost from the genome of the former two

216

strains which was also consistent with the sequence comparison of the draft genomes of

217

BIOMIG1 phenotypes in our previous study17.

218

Both BIOMIG1BAC and BIOMIG1BDMA completely utilized 250 µM BAC within

219

50 h (Figure 2B and C). Given the fact that BDMA transformation rate is faster than

220

BAC conversion to BDMA8, BDMA was not detected in the flasks inoculated with

221

BIOMIG1BAC. Moreover, the cell density increased by approximately 200 folds (Figure

222

2B). The results suggested that BIOMIG1BAC transformed BACs to BDMA which was

223

quickly utilized as a carbon and energy source promoting cell growth. On the other hand,

224

equimolar amount of BDMA was produced when BACs were utilized by BIOMIG1BDMA.

225

However, cell density increased only 10 folds which was less than that of BIOMIG1BAC

226

(Figure 2C) . This results suggested that oxyBAC was only involved in the BAC to


Page 11 of 28


227

BDMA conversion and does not support growth on the BDMA. In contrast, BIOMIG1BD

228

and BIOMIG1N, which did not contain oxyBAC, neither utilized BACs nor grew in the

229

SM containing BACs (Figure 2D and E). In conclusion, absence of oxyBAC in

230

BIOMIG1 resulted in subsequent loss of BAC transformation to BDMA.

231

E. coli overexpressing oxyBAC gene converts BACs to BDMA. In order to further

232

confirm the function of oxyBAC, biotransformation experiments were conducted with E.

233

colioxyBAC cells heterologously expressing the gene. A control assay was prepared with E.

234

coliN cells transformed with an empty plasmid. We verified that oxyBAC was present in

235

E. colioxyBAC and absent in E. coliN using PCR targeting the oxyBAC (Figure 3A). In the

236

biotransformation assay, C12BDMA-Cl was used as the substrate at 20 µM concentration.

237

E. coliN.did not utilize C12BDMA-Cl in the course of incubation (Figure 3B). On

238

the other hand C12BDMA-Cl was completely converted to BDMA within 1.5 h in the

239

flasks inoculated with E. colioxyBAC (Figure 3C). The rate of C12BDMA-Cl

240

biotransformation was predicted as 14 µM hr-1. This results confirmed that oxyBAC is

241

the enzyme responsible for the conversion of BAC to BDMA via N-dealkylation reaction.

242

These rates were very similar with the native enzyme of Pseudomonas sp. BIOMIG117,

243

indicating that oxyBAC could function effectively outside of its original host. The high

244

efficiency in integrating oxyBAC into a heterologous expression system (e.g., E. coli),

245

indicates that the gene might be promiscuous in horizontal gene transfer and easy to

246

manipulate in further biotechnological applications.

247

To date, two other enzymes capable of biotransforming QAC disinfectants have

248

been identified. An amine oxidase of Pseudomonas nitroreducens B13 could convert

249

C12BDMA-Cl and C14BDMA-Cl to BDMA with ~50% efficiency and a rate of 20 nmol



Page 12 of 28

250

substrate/mg of protein/min and 7 nmol substrate mg of protein-1 min-1, respectively and

251

TTABMO of Pseudomonas putida ATCC 12633 could convert

252

tetradecyltrimethylammonium bromide with a rate of 128.6 nmol TMA min-1 mg protein-

253

1

254

enzymes were phylogenetically distant to oxyBAC and groupV ROs. The most closely

255

related enzyme to amine oxidase of Pseudomonas nitroreducens B was a

256

pseudooxynicotine amine oxidase (pao, GenBank accession number: AFD54463.1)14.

257

Both amine oxidases could attact the C-N bond and catalyze a dealkylation reaction.

258

Additionaly, the closest homolog of TTABMO was a typeV secretion protein; Rhs of

259

Pseudomonas sp. (GenBank accession no: WP_049586647.1) These results imply that

260

there might be multiple genetic mechanisms and enzymes capable of biotransforming

261

QAC disinfectants in the environment. Yet, oxyBAC appears to be the most efficient

262

mechanism known, at least based on the product yields in our experiments.

263

oxyBAC is a unique N-dealkylating RO. Rieske type oxygenases are growing group of

264

oxygenase enzymes which are important for biodegradation of xenobiotic pollutants 21, 22.

265

The presence of a conserved Rieske motif (-CXHX15-17CXXH-) and mononuclear iron

266

center qualify oxyBAC as an RO. We demonstrated that oxyBAC cleaves C-N+ bond

267

between a long saturated alkyl chain and a quaternary N, which is a unique reaction type

268

compared to other N-dealkylating ROs, because the other N-dealkylating ROs either

269

targets a methyl group and demethylate an amine 21, 22 or a quaternary ammonium15, 19, or

270

an unsaturated short alkyl chain16. In addition, ROs are multicomponent enzymes,

271

consisting of an oxygenase reductase and sometimes also a ferredoxin subunit 23, 24. In

272

contrast, oxyBAC facilitates without a specific reductase, which suggested that it

. Despite catalyzing similar reactions, those previously identified QAC degrading


Page 13 of 28


273

probably recruits a nonspecific reductase present in the host strain. Although this is

274

uncommon, multicomponent ROs with promiscuous reductases have been identified 25, 26.

275

These facts suggests that oxyBAC is a unique RO.

276

In order to understand the unique features of oxyBAC that gives its novel reaction

277

capabilities, Rieske domain and the mononuclear iron center of oxyBAC was compare to

278

other N-dealkylating ROs (Figure 4). Rieske domain of oxyBAC was 51, 46 and 43%

279

similar to those of cntA, gbcA and stc2, respectively. The key difference between the

280

Rieske domains of oxyBAC and other N-dealkylating ROs was the presence of 19

281

residues, instead of 17 between CXH and CXXH conserved regions. The reason for this

282

was the insertion of two glycine (G) into this region (Figure 4A). In addition, adjacent 2

283

amino acids (LR) before and 3 amino acids (RIL) after the two glycine are unique to

284

oxyBAC where as they are VA and KLV in both stc2 and gbcA (Figure 4A). Thus, LR-

285

GG-RIL motif is unique for oxyBAC Rieske domain and might underlie its unique

286

biochemical function. Moreover, mononuclear iron center of the oxyBAC is 42, 54 and

287

58% similar to those of cntA, gbcA and stc2, respectively. Substitution of bridging

288

aspartate (D) to bridging glutamate (E) is common to all N-dealkylating ROs compared to

289

other ROs including cmo (Figure 4B). Zhu et al (2014) suggested that this substitution is

290

the main reason for the attack on C-N+ in the N-dealkylation reaction which can be

291

achieved by all Group V ROs except cmo which cannot attack on C-N+ bond. On the

292

other hand, substitution of tyrosine (Y) with leucine (L) adjacent to first conserved

293

histidine (H) in mononuclear iron center is unique to oxyBAC (Figure 4B). These unique

294

features of oxyBAC peptide sequence may resulted in the N-dealkylation of saturated



Page 14 of 28

295

long-chain alky groups from QACs without a need of a specific reductase for the reaction.

296

Varification of this hypothesis via genetic experiments should be part of future work.

297 298

ENVIRONMENTAL SIGNIFICANCE AND APPLICATIONS

299

In this study, we identified a gene responsible for detoxification of BACs by converting

300

BACs to BDMA, confirmed its function genetically and also provide a PCR primer

301

specific for the gene. The oxyBAC enzyme can be used to develop advanced wastewater

302

treatment technologies where BACs are problem such as poorly treated wastewater and

303

effluents originating from hospitals and pharmaceutical facilities. In addition, oxyBAC

304

based biotechnology could have applications in bioremediation of environments

305

contaminated with BACs. For instance, the primers developed here can be used to

306

evaluate the BAC treatment potential of activated sludge treating domestic, industrial and

307

agricultural wastewater as well as of the natural environments contaminated with BACs.

308

On the other hand, microorganisms having oxyBAC gene are not desirable in medical

309

environments since they would decrease the efficacy of disinfectant most of which have

310

BACs as active ingredients and thus could cause proliferation of pathogens. Such threats

311

may be minimized by systematically analyzing the abundance of oxyBAC in the medical

312

settings and taking necessary precautions to avoid contamination of indoor environment

313

by microorganisms harboring oxyBAC gene.

314

Protein based experiments are necessary for further characterization of oxyBAC.

315

The multicomponent structure can be fully resolved once the reductase and ferredoxin

316

subunits of the enzyme are identified and analyzed via crystallography. Additionally,

317

Protein characteristics such as substrate specificity and binding efficiency, optimum


Page 15 of 28


318

working temperature, pH, etc. should be determined for implementing technology

319

applications. Also, the stoichiometry of the N-dealkylation reaction needs to be defined

320

by identifying the total repertoire of metabolites. All of these matters are crucial for better

321

understanding of how oxyBAC functions, which is critical for BAC degradation in the

322

environment.

323

The enzymes degrading BACs and QACs are diverse in terms of structure and

324

phylogeny. This suggests that there are possibly a number of enzymes in the environment

325

yet to be discovered. Candidate QAC degrader strains and enzymes can be identified and

326

tested using ongoing whole genome sequencing projects.

327

In summary, the discovery of oxyBAC as a novel RO enzyme expands our

328

knowledge on the biotransformation mechanisms of disinfectants in the environment and

329

applications and complications it provides.

330

331

SUPPORTING INFORMATION

332

oxyBAC amino acid sequence; biotransformation reactions of stachydrine, glycine

333

betaine, carnitine, choline and BACs by stc2, gbcA, cntA, cmo and oxyBAC,

334

respectively; pET29a(+) plasmid map showing the locations of oxyBAC and other genes

335

as well as restriction sites and SDS-PAGE image of crude protein extracts of uninduced

336

and induced E. coli cells with pET29a(+) plasmid with oxyBAC gene. This material is

337

available free of charge via the Internet at http://pubs.acs.org.

338

339

340



341

ACKNOWLEDGEMENT

342

This study was partially supported by Bogazici University Scientific Research Projects

343

Fund (BAP 7130), EU Research Executive Agency FP7-PEOPLE-MC-CIG

344

(ROBODAR-293665), The Scientific and Technological Research Council of Turkey

345

(TUBITAK, 113Y528 and BIDEB 2214a).

Page 16 of 28

346


Page 17 of 28


347

TABLES

348

Table1. Strains, plasmids and primers used in this study Strains, plasmids and primers Pseudomonas sp. BIOMIG1 ::aBAC ::BDMA

Source

A strain isolated from sewage taken from an urban wastewater treatment plant in Istanbul, TR Can utilize both BACs and BDMA Can convert BACs to BDMA but cannot utilize BDMA Cannot utilize BACs but can utilize BDMA Can utilize neither BACs nor BDMA

17

Escherichia coli BL21 (DE3)pLysS ::oxyBAC ::N

Heterologous expression of oxyBAC under the T7 promoter Contains pET29+ with oxyBAC Contains pET29+ with no oxyBAC

Invitrogen

Plasmids pET29a+

For overexpression of oxyBAC

::BD ::N

349

Description

Primers oxyBAC-F 5’ -TTATATGCAACAGATCCATCCCCT-3’ oxyBAC-R 5’-TTATGTACGCCGTAGGCGGGGCC-3’ a appear as superscrips in the text

17 17

17 17

This study This study

Merc Millipore

This study This study

350



Page 18 of 28

351

FIGURES

352

Figure 1. Phylogenetic relationship of oxyBAC, ttmao, BAC amine oxidase, microbial

353

Rieske type terminal oxygenases (Groups I-IV) and eukaryotic Rieske type choline

354

monooxygenases. Benzylsuccinate synthase was used as the outgroup. Amino acid

355

sequences were aligned using CLUSTLW, genetic distances were calculated using Jukes-

356

Cantor model and the tree was built with neighbor-joining model using a bootstrap value

357

of 1000. Reactions show generalized mechanisms of oxidation of substrates by Rieske

358

type oxygenases.

359 360

Figure 2. (A) oxyBAC PCR amplicons of four BIOMIG1 phenotypes on agarose gel.

361

Profile of C12BDMA-Cl utilization, BDMA formation and cell growth by (B)

362

BIOMIG1BAC, (C) BIOMIG1BDMA, (D) BIOMIG1BD and (E) BIOMIG1N (Error bars

363

represent one standard deviation of the means, n = 3).

364 365

Figure 3. (A) oxyBAC PCR amplicons of two E. coli phenotypes on agarose gel. Profile

366

of C12BDMA-Cl utilization and BDMA formation by (B) E. coliN and (C) E. colioxyBAC

367

(Error bars represent one standard deviation of the means, n = 3).

368 369

Figure 4. Multiple sequence alignment of (A) Rieske domain and (B) mononuclear iron

370

center of oxyBAC with other Group V Rieske oxygenases and oxyBAC of

371

Novosphingobium sp. B7. ↑ and ∆ denote amino acid insertion and substitution,

372

respectively.

373


Page 19 of 28


374 375 376

Figure 1. Phylogenetic relationship of oxyBAC, ttmao, BAC amine oxidase, microbial

377

Rieske type terminal oxygenases (Groups I-IV) and eukaryotic Rieske type choline

378

monooxygenases. Benzylsuccinate synthase was used as the outgroup. Amino acid

379

sequences were aligned using CLUSTLW, genetic distances were calculated using Jukes-

380

Cantor model and the tree was built with neighbor-joining model using a bootstrap value

381

of 1000. Reactions show generalized mechanisms of oxidation of substrates by Rieske

382

type oxygenases.

383



Page 20 of 28

384 385

Figure 2. (A) oxyBAC PCR amplicons of four BIOMIG1 phenotypes on agarose gel.

386

Profile of C12BDMA-Cl utilization, BDMA formation and cell growth by (B)

387

BIOMIG1BAC, (C) BIOMIG1BDMA, (D) BIOMIG1BD and (E) BIOMIG1N (Error bars

388

represent one standard deviation of the means, n = 3).

389


Page 21 of 28


390 391

Figure 3. (A) oxyBAC PCR amplicons of two E. coli phenotypes on agarose gel. Profile

392

of C12BDMA-Cl utilization and BDMA formation by (B) E. coliN and (C) E. colioxyBAC

393

(Error bars represent one standard deviation of the means, n = 3).

394



Page 22 of 28

395 396

Figure 4. Multiple sequence alignment of (A) Rieske domain and (B) mononuclear iron

397

center of oxyBAC with other Group V Rieske oxygenases and oxyBAC of

398

Novosphingobium sp. B7. ↑ and ∆ denote amino acid insertion and substitution,

399

respectively.

400


Page 23 of 28

401


GRAPHICAL ABSTRACT

402 403



Page 24 of 28

404

REFERENCES

405

1.

406

Antimicrobial Resistance in the Environment. In Antimicrobial Resistance in the

407

Environment, Keen, P. L.; Montforts, M. H. M. M., Eds. Wiley-Blackwell: New Jersey,

408

2012; pp 349-388.

409

2.

410

Fürhacker, M.; Scharf, S.; Gans, O., Determination of selected quaternary ammonium

411

compounds by liquid chromatography with mass spectrometry. Part I. Application to

412

surface, waste and indirect discharge water samples in Austria. Environ. Pollut. 2007,

413

145, (2), 489-496.

414

3.

415

Milandri, M.; Reinthaler, F.; Verhoef, J., Analysis of benzalkonium chloride in the

416

effluent from European hospitals by solid-phase extraction and high-performance liquid

417

chromatography with post-column ion-pairing and fluorescence detection. J. Chromatogr.

418

A 1997, 774, (1-2), 281-286.

419

4.

420

adaptation, degradation and ecology. Curr. Opin. Biotechnol. 2015, 33, 296-304.

421

5.

422

Perspective. In Handbook of Detergents, Part B., Zoller, U., Ed. CRC Press: New York,

423

2004; pp 523-549.

Tezel, U.; Pavlostathis, S. G., Role of Quaternary Ammonium Compounds on

Martínez-Carballo, E.; Sitka, A.; González-Barreiro, C.; Kreuzinger, N.;

Kümmerer, K.; Eitel, A.; Braun, U.; Hubner, P.; Daschner, F.; Mascart, G.;

Tezel, U.; Pavlostathis, S. G., Quaternary ammonium disinfectants: microbial

Ginkel, C. G. V., Biodegradation of Cationic Surfactants: An Environmental


Page 25 of 28


424

6.

Oh, S.; Tandukar, M.; Pavlostathis, S. G.; Chain, P. S. G.; Konstantinidis, K. T.,

425

Microbial community adaptation to quaternary ammonium biocides as revealed by

426

metagenomics. Environ. Microbiol. 2013, 15, (10), 2850-2864.

427

7.

428

chloride by Aeromonas hydrophila sp. K. J. Appl. Microbiol. 2003, 94, (2), 266-272.

429

8.

430

Aerobic biotransformation of n-tetradecylbenzyldimethylammonium chloride by an

431

enriched Pseudomonas spp. community. Environ. Sci. Technol. 2012, 46, (16), 8714-

432

8722.

433

9.

434

ammonium compounds. Appl. Environ. Microbiol. 1977, 33, (5), 1037-1041.

435

10.

436

Genomic Diversity of Escherichia Isolates from Diverse Habitats. PLoS One 2012, 7,

437

(10).

438

11.

439

Degradation of tetradecyltrimethylammonium by Pseudomonas putida a ATCC 12633

440

restricted by accumulation of trimethylamine is alleviated by addition of Al3+ ions. J.

441

Appl. Microbiol. 2008, 104, (2), 396-402.

442

12.

443

Characterization of Pseudomonas putida A (ATCC 12633) Monooxygenase Enzyme

Patrauchan, M. a.; Oriel, P. J., Degradation of benzyldimethylalkylammonium

Tezel, U.; Tandukar, M.; Martinez, R. J.; Sobecky, P. a.; Pavlostathis, S. G.,

Dean-Raymond, D.; Alexander, M., Bacterial metabolism of quaternary

Oh, S.; Buddenborg, S.; Yoder-Himes, D. R.; Tiedje, J. M.; Konstantinidis, K. T.,

Liffourrena, a. S.; López, F. G.; Salvano, M. a.; Domenech, C. E.; Lucchesi, G. I.,

Liffourrena, A. S.; Lucchesi, G. I., Identification, Cloning and Biochemical



Page 26 of 28

444

necessary for the Metabolism of Tetradecyltrimethylammonium Bromide. Appl. Biochem.

445

Biotechnol. 2014, 173, (2), 552-561.

446

13.

447

M.; Pavlostathis, S. G.; Spain, J. C.; Konstantinidis, K. T., Microbial Community

448

Degradation of Widely Used Quaternary Ammonium Disinfectants. Appl. Environ.

449

Microbiol. 2014, 80, (19), 5892-5900.

450

14.

451

gene from Pseudomonas sp. strain HZN6 whose product nonenantioselectively degrades

452

nicotine to pseudooxynicotine. Appl. Environ. Microbiol. 2013, 79, (7), 2164-71.

453

15.

454

K. N., Quaternary ammonium oxidative demethylation: X-ray crystallographic, resonance

455

raman, and UV-visible spectroscopic analysis of a Rieske-type demethylase. J. Am. Chem.

456

Soc. 2012, 134, (5), 2823-2834.

457

16.

458

Chen, Y., Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase

459

from human microbiota. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, (11), 4268-73.

460

17.

461

and Genes Are Involved in the Biodegradation of Benzalkonium Chlorides in Different

462

Environments. Environ. Sci. Technol. 2016, 50, (8), 4304-4313.

Oh, S.; Kurt, Z.; Tsementzi, D.; Weigand, M. R.; Kim, M.; Hatt, J. K.; Tandukar,

Qiu, J.; Ma, Y.; Zhang, J.; Wen, Y.; Liu, W., Cloning of a novel nicotine oxidase

Daughtry, K. D.; Xiao, Y.; Stoner-Ma, D.; Cho, E.; Orville, A. M.; Liu, P.; Allen,

Zhu, Y.; Jameson, E.; Crosatti, M.; Schäfer, H.; Rajakumar, K.; Bugg, T. D. H.;

Ertekin, E.; Hatt, J. K.; Konstantinidis, K.; Tezel, U., Similar Microbial Consortia


Page 27 of 28


463

18.

Nam, J. W.; Nojiri, H.; Yoshida, T.; Habe, H.; Yamane, H.; Omori, T., New

464

classification system for oxygenase components involved in ring-hydroxylating

465

oxygenations. Biosci. Biotechnol. Biochem. 2001, 65, (2), 254-63.

466

19.

467

M. J.; Brinkman, F. S. L.; Hufnagle, W. O.; Kowalik, D. J.; Lagrou, M.; Garber, R. L.;

468

Goltry, L.; Tolentino, E.; Westbrock-Wadman, S.; Yuan, Y.; Brody, L. L.; Coulter, S. N.;

469

Folger, K. R.; Kas, A.; Larbig, K.; Lim, R.; Smith, K.; Spencer, D.; Wong, G. K. S.; Wu,

470

Z.; Paulsen, I. T.; Reizer, J.; Saier, M. H.; Hancock, R. E. W.; Lory, S.; Olson, M. V.,

471

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic

472

pathogen. Nature 2000, 406, 959-964.

473

20.

474

J.; Scott, P.; Golbeck, J. H.; Hanson, A. D., Choline monooxygenase, an unusual iron-

475

sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants: prosthetic

476

group characterization and cDNA cloning. Proc. Natl. Acad. Sci. U. S. A. 1997, 94, (7),

477

3454-8.

478

21.

479

X.; He, J.; Li, S. P., Novel Three-Component Rieske Non-Heme Iron Oxygenase System

480

Catalyzing the N-Dealkylation of Chloroacetanilide Herbicides in Sphingomonads DC-6

481

and DC-2. Appl. Environ. Microbiol. 2014, 80, (16), 5078-5085.

482

22.

483

P., The Novel Bacterial N-Demethylase PdmAB Is Responsible for the Initial Step of

Stover, C. K.; Pham, X. Q.; Erwin, A. L.; Mizoguchi, S. D.; Warrener, P.; Hickey,

Rathinasabapathi, B.; Burnet, M.; Russell, B. L.; Gage, D. A.; Liao, P. C.; Nye, G.

Chen, Q.; Wang, C. H.; Deng, S. K.; Wu, Y. D.; Li, Y.; Yao, L.; Jiang, J. D.; Yan,

Gu, T.; Zhou, C. Y.; Sorensen, S. R.; Zhang, J.; He, J.; Yu, P. W.; Yan, X.; Li, S.



Page 28 of 28

484

N,N-Dimethyl-Substituted Phenylurea Herbicide Degradation. Appl. Environ. Microbiol.

485

2013, 79, (24), 7846-7856.

486

23.

487

Ramaswamy, S., Structural investigations of the ferredoxin and terminal oxygenase

488

components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1. BMC

489

Struct. Biol. 2007, 7, 10.

490

24.

491

Heme Iron-Dependent Oxygenases. ACS Catal 2013, 3, (10), 2362-2370.

492

25.

493

chemistry and regulation of O2 activation by the oxygenase component of naphthalene

494

1,2-dioxygenase. J. Biol. Chem. 2001, 276, (3), 1945-53.

495

26.

496

Benzoate 1,2-Dioxygenase from Pseudomonas putida: Single Turnover Kinetics and

497

Regulation of a Two-Component Rieske Dioxynase. Biochemistry 2002, 41, 9611-9626.

Ferraro, D. J.; Brown, E. N.; Yu, C. L.; Parales, R. E.; Gibson, D. T.;

Barry, S. M.; Challis, G. L., Mechanism and Catalytic Diversity of Rieske Non-

Wolfe, M. D.; Parales, J. V.; Gibson, D. T.; Lipscomb, J. D., Single turnover

Wolfe, M. D. A. D. J. S., A. ; Popescu C. V. ; Münck E. ; Lipscomb J. D.,

498


A Rieske-Type Oxygenase of Pseudomonas sp. BIOMIG1 Converts

Recommend Documents