Mutation of Phenylalanine-223 to Leucine Enhances Transformation

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Mutation of phenylalanine-223 to leucine enhances transformation of benzo[a]pyrene by ring-hydroxylating dioxygenase of Sphingobium sp. FB3 by increasing accessibility of the catalytic site Bo Fu, Ting Xu, Zhongli Cui, Ho Leung Ng, Kai Wang, Ji Li, and Qing X. Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05018 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Journal of Agricultural and Food Chemistry

Manuscript revised according to editor’s and reviewers’ comments for possible publication in Journal of Agricultural and Food Chemistry

Mutation of phenylalanine-223 to leucine enhances transformation of benzo[a]pyrene by ring-hydroxylating dioxygenase of Sphingobium sp. FB3 by increasing accessibility of the catalytic site

Bo Fu,a,b Ting Xu,a Zhongli Cui,c Ho Leung Ng,d Kai Wang,a Ji Li,a,* Qing X. Lib,**

a

College of Resources and Environmental Sciences, China Agricultural University, 2

Yuanmingyuan West Road, Beijing 100193, China b

Department of Molecular Biosciences and, University of Hawaii at Manoa, Honolulu,

Hawaii 96822, United States c

Department of Microbiology, College of Life Sciences, Key Laboratory for Microbiological

Engineering of Agricultural Environment of Ministry of Agriculture, Nanjing Agricultural University, Nanjing, Jiangsu 201195, China d

Department of Biochemistry & Molecular Biophysics, Kansas State University, Manhattan,

Kansas 66506, United States

Correspondence: *Ji Li Tel: +86 (10) 6273-2017 Fax: +86 (10) 6281-4029 Email: [email protected] **Qing X. Li Tel: (808) 956-2011 Fax: (808) 956-3542 Email: [email protected]

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ABSTRACT

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Burning of agricultural biomass generates polycyclic aromatic hydrocarbons (PAHs) including

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the carcinogen benzo[a]pyrene, of which the catabolism is primarily initiated by a ring-

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hydroxylating dioxygenase (RHD). This study explores catalytic site accessibility and its role in

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preferential catabolism of some PAHs over others. The genes flnA1f, flnA2f, flnA3 and flnA4,

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encoding the oxygenase α and β subunits, ferredoxin, and ferredoxin reductase, respectively, of

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the RHD enzyme complex (FlnA) were cloned from Sphingobium sp. FB3 and co-expressed in E.

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coli BL21. The FlnA effectively transformed fluoranthene, but not benzo[a]pyrene. Substitution

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of the bulky phenylalanine 223 by leucine reduces the steric constraint in the substrate entrance to

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make the catalytic site of FlnA more accessible to large substrates, as visualized by 3D modeling,

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and allows the FlnA mutant to efficiently transform benzo[a]pyrene. Accessibility of the catalytic

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site to PAHs is a mechanism of RHD substrate specificity. The results shed light on why some

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PAHs are more recalcitrant than others.

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KEYWORDS: Biodegradation; Biotransformation; Polycyclic aromatic hydrocarbon; Ring-

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hydroxylating dioxygenase; Sphingobium sp. FB3; Substrate specificity

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INTRODUCTION

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Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, persistent and toxic organic

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pollutants in the environment. Some PAHs such as benzo[a]pyrene are carcinogenic, mutagenic

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and immunosuppressive. PAHs are primarily from incomplete combustion. Field-based

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agricultural biomass burning and wildfires can generate a large amount of PAHs.1-4 PAH-polluted

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soil is a major source of food PAH contamination, posing a serious food safety issue.5-7

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Bioremediation is an effective method to remove PAHs from polluted sites. Many bacterial

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species can degrade low molecular weight (LMW) PAHs.8-10 However, only a small fraction of

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the bacteria isolated thus far can degrade high molecular weight (HMW) PAHs such as

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benzo[a]pyrene.1

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Aerobic biodegradation of PAHs is initiated by introducing two atoms of oxygen to the

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aromatic ring of the substrate.11-14 This reaction, referred to as dioxygenation, is catalyzed by a

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ring-hydroxylating dioxygenase (RHD).15 RHD is a multicomponent bacterial enzyme complex

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consisting of an oxygenase, comprised of an α and β subunit, and an electron transport chain,

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comprised of a ferredoxin and ferredoxin reductase.16 The oxygenase, responsible for

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dioxygenation, catalyzes formation of a cis-dihydrodiol moiety, which is a critical first step to

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bacterial catabolism of various aromatic compounds.17 The electron transport proteins, ferredoxin

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and ferredoxin reductase transfer electrons from an electron donor to the oxygenase component.18

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To date, RHDs related to LMW PAH transformation have been found and validated.19, 20 Very few

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reports, however, demonstrated the function of RHD to dioxygenate HWM PAHs such as

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fluoranthene and benzo[a]pyrene.13

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The α subunit of the oxygenase, which contains a Rieske binding domain and a catalytic domain, is related to substrate specificity.17 Amino acid residues in the distal region of the 3 ACS Paragon Plus Environment


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catalytic site affect the size and shape of substrates that can enter the active site.15 The ring-

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hydroxylating dioxygenase BPDO-ORHA1 from Sphingobium sp. B1 can oxidize benzo[a]pyrene,

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whereas the ring-hydroxylating dioxygenase NDO-O9816-4 from Pseudomonas sp. NCIB 9816-4

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cannot despite that NDO-O9816-4 shares a very similar volume and configuration of the catalytic

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site as BPDO-ORHA1. Based on structural comparison between BPDO-OB1 and NDO-O9816-4,

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Ferraro et al.21 stated “we propose that the shape and size of the active site entrance may keep

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larger substrates out of the NDO-O9816 active site”.

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The present study tested the hypothesis that accessibility of the RHD catalytic site is a

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determining factor for RHD selectivity toward HMW PAHs. A library of mutants of strain

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Sphingobium sp. FB3 was obtained by random Mariner mutagenesis to look for genes related to

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fluoranthene degradation. The genes encoding the RHD complex in Sphingobium sp. FB3, FlnA,

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were cloned and expressed in E. coli BL21. The heterologously expressed FlnA complex was

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active toward fluoranthene, negligible toward benzo[a]pyrene. Computational modeling predicted

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that spatial change of the active site by replacing the large Phe 223 with a small Leu would make

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the site more accessible to benzo[a]pyrene. These predictions were confirmed in vitro wherein the

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mutated complex was active toward benzo[a]pyrene. The results signify that accessibility of the

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catalytic site is a mechanism of RHD substrate specificity.

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MATERIALS AND METHODS

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Reagents. Fluoranthene and benzo[a]pyrene were purchased from Sigma-Aldrich Co.

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(purity>98%) (St. Louis, MO). PAH stock solutions were made in acetone (Beijing Chemical

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Works, Beijing, China) and stored in brown bottles at 4 °C. Fluoranthene was at a concentration

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of 1000 mg/L and benzo[a]pyrene was at 100 mg/L. Antibiotics, isopropyl-β-D4 ACS Paragon Plus Environment

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thiogalactopyranoside and restriction enzymes were purchased from Takara Biotechnology Co.

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(Dalian, Liaoning, China). Primers were obtained from Sangon Biotechnology Co. (Shanghai,

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China).

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Strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study

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were listed in Table 1. Sphingobium sp. FB3 was previously isolated from soil.22 It was grown in

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Lysogeny broth (LB) medium with 50 µg/mL of streptomycin or in mineral salt medium (MSM)

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with PAHs at 30 °C. E. coli strains were grown at 37 °C in LB medium supplemented with

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appropriate antibiotics. Solid media contained 1.5% agar. The concentrations of the antibiotics

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were 50, 50, 50, 50, and 34 µg/mL of ampicillin (Amp), kanamycin (Km), gentamicin (Gm),

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streptomycin (Str), chloramphenicol (Cm), respectively. The MSM contained (per liter) 1.5 g

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K2HPO4, 0.5 g KH2PO4, 1.0 g (NH4)2SO4, 0.03 g MgSO4, and 1.0 g NaCl, pH 7.0.

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Generation of transposon mutants. A mutant library of Sphingobium sp. FB3 was generated to

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screen for genes involved in PAH degradation. Fluoranthene degradation results in formation of

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an orange-yellow metabolite which was used as a selection marker to screen for fluoranthene

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degradation-deficient mutants. The mutant library of strain FB3 was generated by triparental

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conjugation. Sphingobium sp. FB3, E. coli SM10λpir harboring the plasmid pSC123 which

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contained the Mariner transposon,23 and E. coli DH5α harboring the plasmid pRK600 were

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incubated individually in 3 mL of LB medium supplemented with streptomycin, kanamycin,

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chloramphenicol, respectively. Two mL of strain FB3 and 1 mL each of the other two cultures

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were washed thrice with fresh LB medium to remove residual antibiotics, re-suspended in 120 µL

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of LB medium, and combined. The mixture was spotted onto a nitrocellulose filter on an LB plate 5 ACS Paragon Plus Environment


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and incubated at 30 °C for 48 h for conjugation to proceed. The conjugants were re-suspended in

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1 mL of LB medium and spread onto LB plates (10 µL per plate) supplemented with

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streptomycin and kanamycin.24 After incubation at 30 °C for 5 days, all colonies were transferred

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into separate wells on a 96-well plate containing filter-sterilized MSM supplemented with

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fluoranthene.25 Colonies that did not transform fluoranthene were selected for further study.

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Cloning of the sequences flanking the Mariner transposon. The sequences flanking the

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Mariner transposon in the FB3 mutants (5-41 and 12-45) were obtained by thermal asymmetric

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interlaced PCR (TAIL-PCR).26 The primers ARB1, ARB2, ARB3, F-SP1, F-SP2 were used for

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amplifying the 5ʹ flanking sequence of the transposon (Table 2). The first round of amplification

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was performed in a 25 µL reaction volume with 2.5 µL of 10 × PCR buffer, 2.5 µL of 25 mmol/L

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Mg2+, 2 µL of 2.5 mmol/L dNTP mixture, 0.3 µL of 25 µmol/L ARB1, 0.3 µL of 25 µmol/L

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ARB3, 0.8 µL of 25 µmol/L F-SP1, 0.3 µL of 5 U/µL Taq (Takara, Beijing, China). The colony of

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strain FB3 was used as template. The amplifications were performed at 94 °C for 5 min, then 6

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cycles at 94 °C for 30 s, 30 °C for 3 min, 72 °C for 1 min, then another 30 cycles at 94 °C for 30

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s, 55 °C for 30 s, 72 °C for 1 min and finally an extension period of 10 min at 72 °C. The second

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round of amplification was performed in 25 µL reaction volume with 2.5 µL of 10 × PCR buffer,

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2.5 µL of 25 mmol/L Mg2+, 2 µL of 2.5 mmol/L dNTP mixture, 0.5 µL of 25 µmol/L ARB2, 0.5

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µL of 25 µmol/L F-SP2, 0.3 µL of 5 U/µL Taq, and 0.3 µL of the product from the first round of

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TAIL-PCR. The amplifications were performed at 94 °C for 5 min, then 30 cycles at 94 °C for 30

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s, 55 °C for 30 s, 72 °C for 1 min, and finally an extension period of 10 min at 72 °C.

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The PCR products were purified by agarose gel electrophoresis, cloned into the pMD19-T vector (simple) (Takara), then transferred into E. coli DH5α for sequencing to identify the genes 6 ACS Paragon Plus Environment

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that had been disrupted in the mutants. Comparisons of amino acid or nucleotide sequences were

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performed with BLAST on the NCBI website.

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Construction of FlnA expression vectors. Four genes, flnA1f, flnA2f, flnA3 and flnA4, were

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independently amplified by PCR from the genomic DNA of strain FB3 using four primer sets

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(Table 2). The PCR products were purified and separately inserted into the pMD19-T vector

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(simple), sequenced, and then subcloned into the final expression vectors. FlnA1f and flnA2f,

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encoding the α and β subunits of the oxygenase, respectively, were cloned into a single vector

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(pET-A1fA2f), while flnA3 and flnA4, which encode ferredoxin and ferredoxin reductase,

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respectively, were cloned into another vector (pACYC-A3A4). For construction of pET-A1fA2f,

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flnA2f was cloned into the pETDuet-1 vector using restriction sites NcoI and PstI, and flnA1f was

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subsequently cloned into the resulting plasmid using restriction sites NdeI and KpnI. The other

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plasmid, pACYC-A3A4, was similarly constructed by placing flnA4 into the pACYCDuet-1

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vector using restriction sites NcoI and HindIII, and flnA3 was subsequently inserted into the

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resulting plasmid using restriction sites NdeI and KpnI. The resulting plasmids, pET-A1fA2f and

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pACYC-A3A4, were co-transformed into E. coli BL21 (DE3) to form E. coli BL21 FlnA. To

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investigate the function of flnA3 and flnA4, another strain, E. coli BL21 FlnA12, containing pET-

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A1fA2f and pACYCDuet-1 was created. The control was E. coli BL21 CK containing pETDuet-1

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and pACYCDuet-1. The sequence data of flnA1f, flnA2f, flnA3 and flnA4 were submitted to the

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GenBank database under accession numbers MF401193, MF401194, MF401195, and MF401196,

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respectively.

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Residue 223 in the oxygenase α subunit was mutated from Phe to Leu with the overlap extension PCR method using primers A1f223-1-f, A1f223-1-R, A1f223-2-F and A1f223-2-R 7 ACS Paragon Plus Environment


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(Table 2). To construct plasmid pET-A1f223A2f that contains the flnA2f gene (oxygenase β

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subunit) and the mutated flnA1f gene, the amplified PCR product was purified and inserted into

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the pMD19-T vector (simple), sequenced, and then subcloned, using the restriction sites NdeI and

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KpnI, into the pETDuet-1 plasmid already containing the flnA2f gene. Both pET-A1f223A2f and

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pACYC-A3A4 were then co-transformed into E. coli BL21 (DE3) to form E. coli BL21 FlnA223.

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Dioxygenase expression and sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(SDS PAGE). Clones of E. coli BL21 FlnA, E. coli BL21 FlnA12, E. coli BL21 CK, or E. coli

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BL21 FlnA223 were incubated overnight in LB medium at 37 °C. One percent of the culture was

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inoculated into 400 mL LB medium and grown to an OD600 of 0.5 to 0.6. The cultures were

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induced by adding IPTG at a final concentration of 0.5 mM and further incubated for 12 h at 25

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°C. The cells of E. coli BL21 FlnA and E. coli BL21 FlnA12 were harvested, washed, and

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suspended in 10 mL of PBS (pH 7.45), then disrupted by ultrasonication on ice for 10 min (3 s

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interval). The lysates were centrifuged at 15,000g for 10 min. The supernatants were harvested

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and analyzed by SDS-PAGE which was performed on 15% polyacrylamide gels. Protein staining

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was performed with Coomassie brilliant blue R-250.

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PAH transformation by E. coli BL21 FlnA, E. coli BL21 FlnA12, and E. coli BL21 FlnA223.

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After the genes were induced for expression, the cultures of E. coli BL21 FlnA, E. coli BL21

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FlnA12, and E. coli BL21 CK were harvested by centrifugation, washed and resuspended to an

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OD600 of approximately 1.6 in 10 mL MSM containing 100 mg/L fluoranthene. Catalytic

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efficiency of E. coli BL21 FlnA and E. coli BL21 FlnA223 toward benzo[a]pyrene (final

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concentration of 10 mg/L) was investigated in 10 mL MSM with an OD600 of approximately 1.6. 8 ACS Paragon Plus Environment

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The cultures were incubated at 160 rpm for 12 h at 30 °C. All experiments were performed in

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triplicate.

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Extraction and analysis of residual fluoranthene. Residual fluoranthene in the culture was

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extracted with ethyl acetate and analyzed with an Agilent LC1200 high performance liquid

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chromatograph (HPLC) integrated with a diode array detector.22 A venusil MP-C18 column (5

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µm, i.d. 4 mm) was used for separation. The flow rate was 1 mL/min. The gradient was from 60%

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aqueous methanol (HPLC grade) to 100% methanol in the first 30 min, followed by 100%

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methanol for 5 min. PAHs were detected at 250 nm.

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Identification of the fluoranthene metabolite. The metabolite generated in the process of

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fluoranthene degradation by E. coli BL21 FlnA was detected by an Agilent HP 6890N gas

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chromatograph coupled to an Agilent HP 5973 mass selective detector. The samples were run on

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an HP-5MS column (0.25 mm × 30 m × 0.25 µm) with helium as the carrier gas. The initial oven

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temperature was 50 °C, rising 15 °C per minute to a final temperature of 280 °C and kept for 15

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min.

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FlnA1f 3D structure modeling. The 3D structure of FlnA1f was modeled with the Phyre2

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protein fold recognition server.27 The crystal structure of the corresponding α subunit of

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Sphingobium yanoikuyae B1 (2GBX.pdb), sharing 90% amino acid sequence identity to FlnA1f,

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was used as a template.21 Based on the model, the active site of FlnA1f was predicted using the

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PyMOL molecular graphics system by aligning to its template (version 1.6 Schrödinger, LLC).

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The structure of benzo[a]pyrene was drawn and energy minimized with the program Avogadro.28 9 ACS Paragon Plus Environment


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Benzo[a]pyrene was docked into the catalytic site of FlnA1f and FlnA1f223 using AutoDock

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Tools29 and AutoDock Vina 1.1.2.30

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RESULTS AND DISCCUSSION

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Cloning and analysis of the mutant genes from 5-41 and 12-45. A library of mutants of strain

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FB3 was obtained by random Mariner mutagenesis. Two mutants that could not transform

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fluoranthene into orange-yellow color metabolites were screened from a total of 2950 mutants.

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The two mutants that could not degrade fluoranthene (data not shown) were designated as 5-41

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and 12-45. The 5ʹ flanking sequences of the Mariner transposon in 5-41 and 12-45 were amplified

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by TAIL-PCR, and a 129-bp and a 104-bp fragment were obtained, respectively. The disrupted

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genes in mutants 5-41 and 12-45 displayed high similarity to genes encoding the oxygenase α

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subunit and ferredoxin of RHDs reported in other PAH-degrading sphingomonads strains. These

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results indicated involvement of the two genes in the first step of degradation of fluoranthene by

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strain FB3, and were designated as flnA1f and flnA3, respectively.

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Based on genomic DNA information of strain FB3 and the sequence similarities among

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conserved catabolic gene clusters in sphingomonads strains, the gene located immediately

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downstream of flnA1f was predicted to encode the RHD oxygenase β subunit, designated flnA2f

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(data not shown). Ferredoxin always serves as an electron transport chain along with ferredoxin

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reductase.16 Ferredoxin reductase is encoded by a gene 18.3-kb downstream of flnA3 in strain

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FB3, and was designated flnA4. The putative RHD, encoded by flnA1f, flnA2f, flnA3 and flnA4 in

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strain FB3, was designated as FlnA due to its association with fluoranthene degradation.

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Cloning and expression of genes encoding the FB3 RHD. In order to verify the function of 10 ACS Paragon Plus Environment

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flnA1f, flnA2f, flnA3 and flnA4, these four genes were first amplified with corresponding primers.

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They were 1365 bp, 525 bp, 327 bp and 1227 bp in length, respectively. The resulting expression

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plasmids pET-A1fA2f and pACYC-A3A4 were then co-transformed into E. coli BL21 (DE3) to

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produce E. coli BL21 FlnA. To confirm the function of flnA3 and flnA4, E. coli BL21 FlnA12

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was produced to only contain genes flnA1f and flnA2f. E. coli BL21 CK harboring only the

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plasmids without inserted genes was used as a control. As shown in Fig. 1, two polypeptides with

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a size of 50 kDa and 23 kDa induced by IPTG in both E. coli BL21 FlnA and E. coli BL21

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FlnA12 were approximate to the expected values of the α and β subunit, respectively. However,

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no representative bands were seen for the ferredoxin or ferredoxin reductase components, which

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may be attributed to the lower copy number of pACYCDuet-1 which is only a quarter of

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pETDuet-1.19

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Biotransformation of fluoranthene by E. coli BL21 FlnA. RHD has been widely investigated

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because of its importance in PAH degradation.15, 31-33 The biotransformation of fluoranthene by E.

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coli BL21 FlnA and E. coli BL21 FlnA12 was investigated. As shown in Fig. 2, 43% of

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fluoranthene was transformed by E. coli BL21 FlnA within 12 h. The half-life (T1/2) and

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degradation rate constant of fluoranthene by E. coli BL21 FlnA was 0.63 d and 1.1 d-1,

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respectively. The degradation rate constant of fluoranthene by E. coli BL21 FlnA were 3.4-fold

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higher than that by strain FB3.22 The concentrations of residual fluoranthene in the E. coli BL21

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FlnA culture correspondingly decreased over time and one putative metabolite accumulated (Fig.

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3). GC-MS analysis confirmed that the metabolite was monohydroxyfluoranthene (molecular ion

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at m/z 218, retention time 15.408 min) (Fig. 4) which was probably produced by spontaneous

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dehydration of dihydrodiolfluoranthene.13, 31, 34 The results indicated that FlnA catalyzes the 11 ACS Paragon Plus Environment


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initial step of fluoranthene transformation. To date, several RHDs capable of fluoranthene

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degradation were identified in Sphingomonas, but not in Sphingobium.35-37 To our knowledge, the

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present study was the first report to functionally characterize RHD transformation of fluoranthene

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in a strain of the genus Sphingobium.

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Sphingomonads species possess a unique group of genes for aromatic compound

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degradation, which the genes distantly differ from those in Pseudomonas and other genera, both

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in sequence homology and gene organization.38 Although the degradation genes in

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sphingomonads are conserved, subtle structural variations in the α subunits may cause different

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substrate selectivity toward PAHs. The gene phnA1a, encoding the α subunit of RHD in

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Sphingomonas paucimobilis EPA505, shares 99% identity with flnA1f in strain FB3. However,

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when the four genes (phnA1a, phnA2a, phnA3 and phnA4) encoding the RHD PhnI in strain

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EPA505 were expressed in E. coli BL21 (DE3), no fluoranthene transformation was observed.19

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Strain FB3 can utilize and grow with fluoranthene as the sole carbon source, whereas

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Sphingobium yanoikuyae B1 can only co-oxidize fluoranthene, but cannot support growth despite

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that the α subunits of dioxygenases from the two strains sharing 90% identity.39

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As shown in Fig. 2, no degradation of fluoranthene was observed in E. coli BL21 FlnA12

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culture and E. coli BL21 CK. This confirmed that the genes flnA3 (encoding ferredoxin) and

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flnA4 (encoding ferredoxin reductase) are necessary for FlnA in strain FB3 to transform

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fluoranthene into intermediate metabolites. Ferredoxin and ferredoxin reductase are components

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of electron transport chain (ETC) in RHDs. The ETC is required for the RHD to transfer electrons

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from the electron donor to an aromatic hydrocarbon electron acceptor.16 In most cases, expression

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of genes encoding ETC components along with oxygenase is required to catalyze PAHs

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transformation. When BL21 (DE3) (pD12) cells expressing only phnA1a and phnA2a were grown 12 ACS Paragon Plus Environment

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in the presence of PAHs, no dihydrodiol product was detected by GC-MS.19 Clones containing

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the genes encoding the oxygenase alone from Sphingobium yanoikuyae B1 did not catalyze

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transformation of biphenyl or naphthalene.39 In contrast, the nidAB dioxygenase genes of

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Mycobacterium vanbaalenii PYR-1 were functional in an E. coli system despite the absence of

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the ferredoxin and reductase components.40 It was speculated that the oxygenase component of

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RHD borrowed the ferredoxin and reductase components of another electron transport system in

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the recombinant E. coli cells.41 E. coli cells expressing only the RHD oxygenase components,

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phnA1a and phnA2a, from strain CHY-1 could transform phenanthrene into dihydrodiol products.

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However, the catalytic activity was enhanced by 35 times when phnA3 and phnA4 encoding

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ferredoxin and reductase were expressed along with phnA1a and phnA2a.42 Therefore, co-

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expression of [3Fe-4S] type ferredoxin and reductase genes are required for a fully functional

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RHD.

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Benzo[a]pyrene docking to the catalytic site of FlnA1f. RHD is one of the most important

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enzymes in PAH degradation. The oxygenase α subunit is related to substrate selectivity. Only

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negligible degradation of benzo[a]pyrene (6 ± 2% in 10 days) was achieved by strain FB3.22 We

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hypothesized that degradation was constrained due to the small size of the catalytic site relative to

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the large five-ring system, benzo[a]pyrene. The 3D structure of FlnA1f was constructed using

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homology modeling on the basis of the crystal structure of the corresponding α subunit from

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Sphingobium yanoikuyae B1.21 Benzo[a]pyrene was docked into the catalytic site of FlnA1f. The

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results showed that the FlnA1f catalytic site is large enough to well accommodate benzo[a]pyrene

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molecule. This suggested that there must be other reasons rather than steric constraint resulting in

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negligible degradation of benzo[a]pyrene by strain FB3. 13 ACS Paragon Plus Environment


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Effect of mutation on benzo[a]pyrene transformation. The loops which cover the entrance of

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the catalytic site of RHD of strain CHY-1 possibly control the substrate’s access to the catalytic

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site.15 Leu223 on loop 1 and Ile260 on loop 2 contributed to the selectivity of the catalytic site

275

toward HMW PAHs.15 In FlnA1f, position 223 is Phe, which is much larger than Leu (Fig. 5A

276

and B). It seems that Phe223 occupies approximately half of the entrance (Fig. 5A). This would

277

impede benzo[a]pyrene from entering into the catalytic site. Therefore, substitution of Phe223 by

278

Leu223 in FlnA1f formed FlnA223. E. coli BL21 FlnA223 was used to investigate the catalytic

279

activity toward benzo[a]pyrene. The transformation rate of benzo[a]pyrene by E. coli BL21

280

FlnA223 was greater than 5 times faster than that by E. coli BL21 FlnA within 12 h (Fig. 5C).

281

The results demonstrated that Phe223 is a critical residue preventing benzo[a]pyrene from

282

entering the catalytic site for transformation. The transformation rate could be enhanced by

283

substitution of Phe223 with a smaller amino acid residue, such as leucine.

284

Many bacteria are capable of degrading LMW PAH, such as naphthalene, phenanthrene, and

285

anthracene. However, only a few isolates were reported to degrade HMW PAHs, such as

286

benzo[a]pyrene. The effects of residue substitution on the catalytic activity to PAHs in the active

287

site have been investigated by others.17, 43, 44 However, the effect of substitutions outside of the

288

active site was less investigated.45 Our study sheds light on understanding structural determinants

289

of RHD substrate specificity toward PAHs. Furthermore, the engineered FlnA223 potentially

290

provide applications on HMW PAHs degradation.

291

Pellequer et al. 46 first reported that the π-cation interaction between a cationic amino acid

292

residue and a neutral aromatic substrate can stabilize the substrate binding. The phenyl ring of

293

polychlorinated biphenyls (PCBs) contacts the guanidinium group of ArgL46, also creating a π14 ACS Paragon Plus Environment

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294

cation interaction by which this cationic group would stabilize the bound aromatic substrate.47

295

Similarly, π-π stacking can occur between an aromatic substrate and an aromatic amino acid

296

residue. In FlnA1f, π-π stacking between aromatic ring of Phe223 and benzo[a]pyrene may

297

inhibit the innerward movement of benzo[a]pyrene, thus impede it entering the active site. It is

298

possible that substitution of Phe223 by Leu223 prevents π-π stacking, which would allow

299

benzo[a]pyrene pass the entrance to the active site for catalysis by FlnA. Pellequer et al.47

300

indicated that high selectivity of the monoclonal antibody variable fragment S2B1 to coplanar

301

PCBs was due to the steric constraint of ortho chlorines with TrpH33 and TrpH98 in the binding

302

site.

303

The present study was the first report of an RHD (FlnA) in the genus Sphingobium catalyzing

304

transformation of fluoranthene. The mutant E. coli BL21 FlnA223 that increased transformation

305

efficiency of benzo[a]pyrene has approved that accessibility of HMW PAHs into the catalysis site

306

of RHDs is a determining factor of the RHD catalysis selectivity. It was confirmed that the less

307

bulky amino acid residue Leu223 improved the accessibility of benzo[a]pyrene to the catalytic

308

site and further improved the catalytic efficiency. The finding of this study is valuable to

309

strategize bioremediation approaches for the sites contaminated with HMW PAHs and aromatic

310

pesticides where dioxygenation is involved. It also helps to explain profiles of PAHs and aromatic

311

compounds in weathered contaminated sites.

312 313

ABBREVIATIONS

314

Amp, ampicillin; BaP, benzo[a]pyrene; Cm, chloramphenicol; ETC, electron transport chain; Gm,

315

gentamicin; HMW, high molecular weight; Km, kanamycin; LB, Lysogeny broth; LMW, low

316

molecular weight; MSM, mineral salt medium; PAHs, polycyclic aromatic hydrocarbons; PCB, 15 ACS Paragon Plus Environment


317

polychlorinated biphenyl; RHD, ring-hydroxylating dioxygenase; Str, streptomycin

318 319

ACKNOWLEDGMENT

320

We are grateful to Dr. Yanjun Xu of China Agricultural University for the analysis of metabolites

321

and Dr. Margaret R. Baker of the University of Hawaii at Manoa for helpful discussion.

322

FUNDING

323

This work was in part supported by the National Science and Technology Supporting Research

324

Program from MOST, China (2012BAD14B01); the US National Institutes of Health Research

325

Centers in Minority Institutions Program (8 G12 MD007601), and the US National Science

326

Foundation Career Award (1350555). BF was a China Agricultural University scholarship

327

recipient.

328 329

NOTES

330

The authors declare no conflict of interest.


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332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374

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FIGURE CAPTIONS Fig. 1. SDS-PAGE image of crude extracts of FlnA and FlnA12. The α subunit and β subunit of RHD FlnA and FlnA12, respectively, are indicated by arrows. Fig. 2. Comparison of degradation kinetics of fluoranthene by E. coli BL21 FlnA, E. coli BL21 FlnA12, and E. coli BL21 CK. Fig. 3. HPLC chromatograms of fluoranthene (retention time: 28.9 min) and a metabolite (13.1 min) in E. coli BL21 FlnA culture extracts after 6 and 12 hours. Fig. 4. Total ion chromatogram and mass spectrum of metabolites produced by fluoranthene conversion in E. coli BL21 FlnA culture extracts. Fig. 5. (A) (B) Horizontal view of ring-hydroxylating dioxygenase α subunit facing the substrate entrance. The surfaces of substrate catalytic site are shown in grey. Benzo[a]pyrene is shown in red. Loops LI and LII that form the entrance of substrate binding site are shown in cyan and green, respectively. Amino acid residues Phe223 and Leu223 are shown in cyan. This substrate entrance view shows the importance of amino acid residue 223 on accessibility of benzo[a]pyrene to the substrate catalytic site. (C) Camparison of catalytic efficiencies of benzo[a]pyrene by E. coli BL21 FlnA (FlnA), E. coli BL21 FlnA223 (FlnA223), and no bacteria control (ck)



Fig. 1. SDS-PAGE image of crude extracts of FlnA and FlnA12. The α subunit and β subunit of RHD FlnA and FlnA12, respectively, are indicated by arrows.


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Fig. 2. Comparison of degradation kinetics of fluoranthene by E. coli BL21 FlnA, E. coli BL21 FlnA12 and E. coli BL21 CK.


Relative peak intensity


Retention time (min) Fig. 3. HPLC chromatograms of fluoranthene (retention time: 28.9 min) and a metabolite (13.1 min) in E. coli BL21 FlnA culture extracts after incubation of 6 and 12 h.


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Fig. 4. GC-MS total ion chromatogram (A) and mass spectrum (B) of metabolites produced by fluoranthene transformation in E. coli BL21 FlnA culture extracts



Fig. 5. (A) (B) Horizontal view of ring-hydroxylating dioxygenase α subunit facing the substrate entrance. The surfaces of substrate catalytic site are shown in grey surface. Benzo[a]pyrene is shown in red sticks. Loops LI and LII that form the entrance of substrate binding site are shown in cyan and green, respectively. Amino acid residues Phe223 and Leu223 are shown in cyan. This substrate entrance view shows the importance of amino acid residue 223 on accessibility of BaP to substrate catalytic site. (C) Camparison of catalytic efficiencies of benzo[a]pyrene by E. coli BL21 FlnA (FlnA), E. coli BL21 FlnA223 (FlnA223), and no bacteria control (ck).


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Table 1. Bacterial strains and plasmids used in this study plasmids or strains

relevant characteristics

plasmids pSC123

Kmr, vector for transposon-mediated mutagenesis of

pRK600

bacteria Gmr, helper plasmid, mob+, tra+

pMD19-T (simple)

T-A cloning vector, Ampr

pETDuet-1

Ampr, expression vector

pACYCDuet-1

Cmr, expression vector

pET-A1fA2f

pETDuet-1 containing flnA1f and flnA2f

pACYC-A3A4

pACYCDuet-1contaning flnA3 and flnA4

pET-A1f223A2f

pETDuet-1 containing flnA1f223 and flnA2f

strains Sphingobium sp. FB3

PAH degrading isolated

Spningobium sp. 5-41

mutant of FB3 inserting by transpose mariner

Spningobium sp. 12-45

mutant of FB3 inserting by transpose mariner

E. coli DH5α

host strain for cloning vectors

E. coli BL21 (DE3)

F_ompT hsdS(rB- mB-) gal dcm lacY1

E. coli SM10λpir

conjugation strain

E. coli BL21 FlnA

containing plasmids pET-A1fA2f and pACYC-A3A4

E. coli BL21 FlnA12

containing plasmids pET-A1fA2f and pACYCDuet-1

E. coli BL21 CK

containing plasmids pETDuet-1 and pACYCDuet-1

E. coli BL21 FlnA223

containing plasmids pET-A1f223A2f and pACYC-A3A4



Table 2. Oligonucleotide primers used in this study primers

sequences (5’-3’)a

purposes

A1f-F

CATATGAGCGGCGACACCACACTC

amplification of flnA1f

A1f-R

GGTACCTCATTCGGCCGCGTTGAG


A2f-F

CCATGGGCATGTCGACCGAACAAG


A2f-R

CTGCAGCTAACAAAAGAAATACAGATTC


A3-F

CATATGTCGAACAAACTGCGCCTTT

amplification of flnA3

A3-R

GGTACCTCAGGCGCTCCCTTCCG


A4-F

CCATGGGCGTGCGCTCGATTGCT


A4-R

AAGCTTTCAGCCCGCCTGCTTGA


A1f223-1-F

CATATGAGCGGCGACACCACACTC

amplification of flnA1f223

A1f223-1-R

GCCAGTCCCGCCAACGGACC


A1f223-2-F

GGTCCGTTGGCGGGACTGGC


A1f223-2-R

GGTACCTCATTCGGCCGCGTTGAG


ARB1

GGCCACGCGTCGACTAGTACNNNNNNNN

amplification of 5’ flanking

NNGATAT

sequence of the transposon

GGCCACGCGTCGACTAGTACNNNNNNNN


NNACGCC

sequence of the transposon

GGCCACGCGTCGACTAGTAC


ARB2 ARB3

sequence of the transposon F-SP1

AGCCAGGGATGTAACGCACT

amplification of 5’ flanking sequence of the transposon

F-SP2

TAACGGCTGACATGGGAATT

amplification of 5’ flanking sequence of the transposon

a

Restriction sites of primers are italic.


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TABLE OF CONTENT GRAPHIC


Mutation of Phenylalanine-223 to Leucine Enhances Transformation

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