THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 280, No. 26, Issue of July 1, pp. 24759 –24767, 2005
`Printed in U.S.A.
`
`A Three-component Dicamba O-Demethylase from
`Pseudomonas maltophilia, Strain DI-6
`GENE ISOLATION, CHARACTERIZATION, AND HETEROLOGOUS EXPRESSION*
`
`Received for publication, January 18, 2005, and in revised form, March 16, 2005
`Published, JBC Papers in Press, April 26, 2005, DOI 10.1074/jbc.M500597200
`
`Patricia L. Herman‡, Mark Behrens, Sarbani Chakraborty, Brenda M. Chrastil, Joseph Barycki,
`and Donald P. Weeks§
`From the Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 65888-0664
`
`Dicamba O-demethylase is a multicomponent enzyme
`from Pseudomonas maltophilia, strain DI-6, that cata-
`lyzes the conversion of the widely used herbicide di-
`camba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA
`(3,6-dichlorosalicylic acid). We recently described the
`biochemical characteristics of the three components of
`this enzyme (i.e. reductaseDIC, ferredoxinDIC, and oxy-
`genaseDIC) and classified the oxygenase component of
`dicamba O-demethylase as a member of the Rieske non-
`heme iron family of oxygenases. In the current study, we
`used N-terminal and internal amino acid sequence in-
`formation from the purified proteins to clone the genes
`that encode dicamba O-demethylase. Two reductase
`genes (ddmA1 and ddmA2) with predicted amino acid
`sequences of 408 and 409 residues were identified. The
`open reading frames encode 43.7- and 43.9-kDa proteins
`that are 99.3% identical to each other and homologous to
`members of the FAD-dependent pyridine nucleotide re-
`ductase family. The ferredoxin coding sequence (ddmB)
`specifies an 11.4-kDa protein composed of 105 residues
`with similarity to the adrenodoxin family of [2Fe-2S]
`bacterial ferredoxins. The oxygenase gene (ddmC) en-
`codes a 37.3-kDa protein composed of 339 amino acids
`that is homologous to members of the Phthalate family
`of Rieske non-heme iron oxygenases that function as
`monooxygenases. Southern analysis localized the oxy-
`genase gene to a megaplasmid in cells of P. maltophilia.
`Mixtures of the three highly purified recombinant di-
`camba O-demethylase components overexpressed in
`Escherichia coli converted dicamba to DCSA with an
`efficiency similar to that of the native enzyme, suggest-
`ing that all of the components required for optimal en-
`zymatic activity have been identified. Computer model-
`ing suggests that oxygenaseDIC has strong similarities
`with the core ␣ subunits of naphthalene 1,2-dioxygen-
`ase. Nonetheless, the present studies point to dicamba
`O-demethylase as an enzyme system with its own unique
`combination of characteristics.
`
`* This work was supported by funds from United AgriProducts, Inc.
`and the Consortium for Plant Biotechnology Research, Inc. This is
`University of Nebraska Agricultural Research Division journal series
`number 14702. The costs of publication of this article were defrayed in
`part by the payment of page charges. This article must therefore be
`hereby marked “advertisement” in accordance with 18 U.S.C. Section
`1734 solely to indicate this fact.
`The nucleotide sequence(s) reported in this paper has been submitted
`to the GenBankTM/EBI Data Bank with accession number(s) AY786442,
`AY786443, AY786444, and AY786445.
`‡ Present address: School of Biological Sciences, University of Ne-
`braska-Lincoln, Lincoln, NE 68588-0118.
`§ To whom correspondence should be addressed: Dept. of Biochemis-
`try, University of Nebraska-Lincoln, N158 Beadle Center, Lincoln, NE
`68588-0664. Tel.: 402-472-7917; Fax: 402-472-7842; E-mail: dweeks@
`unlnotes.unl.edu.
`
`The herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid)
`has been used to effectively control broadleaf weeds in crops
`such as corn and wheat for almost 40 years. Like a number of
`other chlorinated organic compounds, dicamba does not persist
`in the soil because it is efficiently metabolized by a consortium
`of soil bacteria under both aerobic and anaerobic conditions
`(1– 4). Studies with different soil types treated with dicamba
`have demonstrated that 3,6-dichlorosalicylic acid (DCSA),1 a
`compound without herbicidal activity, is a major product of the
`microbial degradation process (2, 3, 5). Soil samples taken from
`a single site exposed to dicamba for several years yielded a
`number of bacterial species capable of utilizing dicamba as a
`sole carbon source (6). These soil microorganisms could com-
`pletely mineralize dicamba to carbon dioxide, water, and chlo-
`ride ion (7). Studies on the metabolism of dicamba in the cells
`of one of these bacteria, the DI-6 strain of Pseudomonas mal-
`tophilia, showed that DCSA is a major degradation product
`(7, 8).
`We have been investigating dicamba O-demethylase, the
`enzyme involved in the first step of the dicamba degradation
`pathway in P. maltophilia, strain DI-6. We previously demon-
`strated that cell lysates contain an O-demethylase that cata-
`lyzes the rapid conversion of dicamba to DCSA (9). We also
`partially purified the enzyme and found that at least three
`separate components are required for activity (9). Recently, we
`provided a detailed description of the purification and charac-
`terization of the reductaseDIC, ferredoxinDIC, and oxygenaseDIC
`components of dicamba O-demethylase (10). OxygenaseDIC is a
`homotrimer (␣)3 with a subunit molecular mass of ⬃40 kDa
`and contains a single Rieske [2Fe-2S] cluster. FerredoxinDIC is
`a monomer with an estimated molecular mass of 14 kDa and
`has a single [2Fe-2S] cluster resembling those found in adreno-
`doxin and putidaredoxin. ReductaseDIC, a monomer with a
`molecular mass of ⬃45 kDa, has the typical yellow color and
`UV fluorescence indicative of a flavin-containing molecule. All
`of the biochemical and physical data suggest that oxygenaseDIC
`can be classified as a member of the family of Rieske non-heme
`iron oxygenases (11).
`In the present study, we describe the cloning and character-
`ization of the genes (designated as ddmA, ddmB, and ddmC)
`that encode the three components of dicamba O-demethylase
`from P. maltophilia, strain DI-6. We demonstrate by Southern
`analysis that the oxygenase gene (ddmC) can be localized to a
`megaplasmid in cells of P. maltophilia. Finally, we describe
`overexpression of each of the cloned genes in a heterologous
`system and demonstrate that the three purified recombinant
`components can be reconstituted into an active enzyme that
`
`This paper is available on line at http://www.jbc.org
`
`This is an Open Access article under the CC BY license.
`
`1 The abbreviations used are: DCSA, 3,6-dichlorosalicylic acid; DIC,
`dicamba; HPLC, high performance liquid chromatography; DIG, digoxi-
`genin; ORF, open reading frame.
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`Dicamba O-Demethylase Genes
`
`TABLE I
`Amino acid sequences, PCR primers, and oligonucleotide used
`in the cloning of the genes encoding the three components
`of dicamba O-demethylase
`The sequence of the degenerate nested PCR primers and oligonucleo-
`tide was based on the underlined amino acid residues.
`
`Component
`
`Sequence
`
`Reductase
`N-terminal
`Internal
`
`Nested PCR primers
`A
`B
`C
`PCR primers (probe)
`
`Ferredoxin
`N-terminal
`Consensus
`
`Nested PCR primers
`A
`B
`C
`PCR primers (probe)
`
`SKADVVIVGAGHGGAQ(C)AIALQN
`LYIRPPTFWA
`
`5⬘-AARGCNGAYGTNGTNAT-3⬘
`5⬘-ATHGTNGGNGCNGGNCA-3⬘
`5⬘-GTNGGNGGNCKDATRTA-3⬘
`5⬘-GGGCATGGCGGTGCACA-3⬘
`5⬘-AGGCGGTCGAAGGTCTT-3⬘
`
`PQITVVNQSGEESSVEASEGRTLMEVIRD
`RL(T/S/C)CQ(V/I/L)
`
`5⬘-ATHACNGTNGTNAAYCA-3⬘
`5⬘-ATGGARGTNATHMGNGA-3⬘
`5⬘-ANYTGRCANSWNARNCG-3⬘
`5⬘-ATGGAGGTTATTCGCGACA-3⬘
`5⬘-GCTGTCGAGCAGGTCGTTC-3⬘
`
`Oxygenase
`N-terminal
`Oligonucleotide (probe)
`
`TFVRNAWYVAALPEELSEKPLGRTILD
`5⬘-AAYGCNTGGTAYGTSGC-3⬘
`
`can convert dicamba to DCSA with an efficiency similar to that
`observed for the native enzyme under our assay conditions.
`
`EXPERIMENTAL PROCEDURES
`Bacterial Strain and Culture Medium—P. maltophilia, strain DI-6,
`was originally isolated from a soil sample collected near a storm water
`retention pond at a dicamba manufacturing plant in Beaumont, TX (6).
`Cells were grown in reduced chloride medium (6) amended with either
`filter-sterilized 5 mM dicamba or with autoclaved glucose (2 mg/ml) and
`casamino acids (2 mg/ml) as the carbon source. Solidified medium was
`prepared with 1% (w/v) Gelrite.
`Materials—Dicamba was a generous gift from Sandoz Agro Inc. (Des
`Plaines, IL). The custom oligonucleotide primers utilized in this study
`were commercially synthesized by Operon (Alameda, CA) and are listed
`in Table I.
`Isolation of Genomic DNA—Genomic DNA was isolated from P. mal-
`tophilia according to a protocol modified from a method used for Syn-
`echococcus 6301 in the laboratory of Donald Bryant at Penn State
`University.2 Cells were grown in 500 ml of reduced chloride medium
`with glucose and casamino acids at 30 °C to an A600 of 1.5–2.0 and
`harvested by centrifugation at 9,110 ⫻ g for 20 min. The cells in the
`pellet were resuspended in sucrose buffer (50 mM Tris (pH 7.5), 10 mM
`EDTA, 10% sucrose), incubated with lysozyme (5 mg/ml) for 30 min at
`37 °C, and then lysed in 1% Sarkosyl. The lysate was centrifuged to
`equilibrium in a CsCl-ethidium bromide gradient in a Type 90 Ti rotor
`(Beckman) at 214,200 ⫻ g for 72 h at 20 °C. The fraction containing the
`genomic DNA was extracted with n-butanol and precipitated with 0.3 M
`sodium acetate and ethanol.
`Isolation of Megaplasmid DNA—Cells of P. maltophilia were grown
`in 500 ml of reduced chloride medium with 5 mM dicamba for ⬃48 h at
`30 °C with shaking (225 rpm). At this point, it was necessary to replace
`the culture medium because a metabolic by-product that interferes with
`cell growth typically accumulates in cultures of P. maltophilia grown
`with dicamba as the sole carbon source. The culture was centrifuged
`under sterile conditions at 5,000 ⫻ g for 10 min and then the pellet was
`resuspended in 500 ml of fresh or reduced chloride medium with 5 mM
`dicamba. The culture was grown for another 72 h under the same
`conditions and then plasmid DNA was isolated from the cells with a
`Qiagen-tip 100 according to a protocol recommended by the manufac-
`turer (Qiagen) for the purification of very low-copy plasmids.
`Amino Acid Sequencing—The purification of the reductaseDIC, ferre-
`
`2 D. Bryant, personal communication.
`
`doxinDIC, and oxygenaseDIC components of dicamba O-demethylase as
`well as the N-terminal amino acid sequences of the purified proteins
`have been described (10). To obtain internal amino acid sequence in-
`formation, the purified ferredoxinDIC and reductaseDIC proteins were
`digested with trypsin and the isolated peptide fragments were se-
`quenced by automated Edman degradation in the Protein Core Facility
`(Center for Biotechnology, University of Nebraska-Lincoln).
`PCR Amplification and Cloning—A PerkinElmer DNA Thermal Cy-
`cler (model 480) programmed with the following profile was used for most
`PCR reactions: 97 °C for 5 min; 30 cycles of 95 °C for 1 min, 55 °C for 1
`min, 72 °C for 2 min, and 72 °C for 7 min. Reaction mixtures (50 ␮l)
`typically contained 5 ␮l of 10 times buffer (Invitrogen), 1.5 mM MgCl2,
`0.1% Triton X-100, 200 ␮M dNTPs, 100 pmol of each primer, 10–100 ng of
`template DNA, and 2.5 units of Taq polymerase (Invitrogen). Pfu polym-
`erase (2.5 units) (Stratagene) was used in PCR in which new restriction
`sites were added to the gene coding regions to facilitate cloning into a pET
`expression vector. Amplified products were ligated into the vector
`pGEM-T Easy (Promega) or pBluescript II KS⫹ (Stratagene) and the
`mixture was transformed into competent Escherichia coli DH5␣ cells.
`Plasmid DNA was isolated from selected bacterial colonies with a QIA-
`prep Spin Miniprep kit according to the manufacturer’s protocol (Qiagen).
`Clones were screened using standard molecular techniques that included
`appropriate restriction digests and agarose gel electrophoresis (12). Both
`strands of selected clones were sequenced by the Genomics Core Research
`Facility (Center for Biotechnology, University of Nebraska-Lincoln) using
`standard sequencing primers.
`Preparation and Screening of Size-fractionated Genomic Libraries—
`Genomic DNA from P. maltophilia (at least 10 ␮g) was digested with
`appropriate restriction enzymes and resolved on a 1% agarose gel using
`standard molecular techniques (12). Gel pieces containing restriction
`fragments of the desired size were excised and digested with ␤-agarase
`according to the protocol recommended by the manufacturer (New
`England Biolabs). The DNA fragments eluted from the gel were ligated
`into the vector pBluescript II KS⫹ (Stratagene) and the mixture was
`transformed into competent E. coli DH5␣ cells. Colony lifts were pre-
`pared from each library and the bacterial clones were screened with
`gene-specific digoxigenin (DIG)-labeled probes as described below. Plas-
`mid DNA from positive bacterial colonies was isolated, characterized,
`and sequenced as described in the preceding section.
`Southern Blots, and Colony Lifts, and Probes—Blots and colony lifts
`were prepared and hybridized with probes labeled with DIG according
`to the standard protocols in the DIG Application Manual (Roche). Dou-
`ble-stranded DNA probes were labeled with DIG-11-dUTP using either
`a standard PCR or a random primed method detailed in the DIG
`Application Manual. Oligonucleotide probes were labeled with a DIG
`Oligonucleotide 3⬘-end Labeling Kit (Roche). Nylon filters were always
`washed under very stringent conditions after hybridization, that is,
`twice for 10 min at 65 °C in 2⫻ SSC with 0.1% SDS and then twice for
`20 min at 65 °C in 0.1⫻ SSC with 0.1% SDS. DIG-labeled DNA was
`detected by the chemiluminescent substrate CSPD according to the
`protocol recommended by the manufacturer (Roche).
`Cloning of the Reductase Genes—The first 23 residues of the N-
`terminal sequence was determined for the purified reductaseDIC protein
`(Table I) as described previously (10). A comparison of this sequence to
`the GenBankTM data base showed that it was 90% identical in a 20-
`amino acid overlap to the cytochrome P450-type reductase component
`of dioxin dioxygenase, a three-component enzyme previously isolated
`from Sphingomonas sp. RW1 (13). An internal sequence of 10 residues
`obtained from tryptic digests of the purified reductaseDIC protein also
`was 80% identical to residues 61 through 70 of the same Sphingomonas
`reductase. This sequence information was used to clone the reductase
`gene by a two-step nested PCR approach. Three degenerate oligonu-
`cleotide primers (two sense and one antisense) were designed and
`synthesized (Table I). The sequence of the two sense primers was based
`on the N-terminal amino acid sequence of the purified reductaseDIC.
`Primer A (17-mer, 256 variants) was based on the sequence KADVVI
`and primer B (17-mer, 768 variants) was derived from the sequence
`IVGAGH. The sequence of the antisense primer C (17-mer, 768 vari-
`ants) was based on the internal sequence YIRPPTF. Primers A and C
`were used in a PCR with P. maltophilia genomic DNA as template. An
`aliquot containing a mixture of the products from the first PCR was
`then used as the template in a second round of amplification with
`primers B and C. A 180-bp product was amplified in the second PCR
`and sequenced. The amino acid sequence predicted by this clone
`matched the N-terminal and internal sequence from the purified reduc-
`taseDIC protein. New sense and antisense primers based on the DNA
`sequence of the clone were designed (Table I) and a 148-bp probe was
`labeled in a PCR. The DIG-labeled probe consistently detected two
`
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`Dicamba O-Demethylase Genes
`
`24761
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`fragments of different sizes when it was hybridized at 68 °C under very
`stringent conditions to several restriction digests of P. maltophilia
`genomic DNA that had been blotted to a nylon membrane (data not
`shown). This result suggested that there are two reductase genes lo-
`cated at different loci in the genome of P. maltophilia. A map of the
`restriction sites surrounding the reductase genes was constructed
`based on the sizes of the various restriction fragments that hybridized
`to the probe. This restriction map suggested that full-length copies of
`the two reductase genes were contained on 4- and 20-kb KpnI/EcoRI
`fragments. To clone the first gene, a size-fractionated genomic library
`containing 3.0 –5.0-kb KpnI/EcoRI fragments was constructed and col-
`ony lifts were prepared. The 148-bp reductase probe was hybridized to
`⬃200 bacterial clones from the library at 68 °C and one positive clone
`was selected. To clone the second gene, KpnI/EcoRI fragments of
`P. maltophilia genomic DNA with a size of 15–25 kb were gel purified,
`digested with a number of restriction enzymes, and then hybridized by
`Southern blot to the same reductase probe. A second restriction map,
`constructed according to the sizes of restriction fragments that hybrid-
`ized to the probe, suggested that a full-length reductase gene was
`contained on a 3.0-kb ApaI fragment. Subsequently, a size-fractionated
`genomic library containing 2.0 – 4.0-kb ApaI fragments was constructed
`and colony lifts were prepared. The reductase probe was hybridized to
`⬃200 bacterial clones from the library at 68 °C and one positive clone
`was selected.
`Cloning of the Ferredoxin Gene—The first 29 residues of the N-
`terminal amino acid sequence was determined for the purified ferre-
`doxinDIC protein (Table I) as described previously (10). A comparison of
`this sequence to the GenBank data base showed that it was 35%
`identical in a 26-amino acid overlap to a terpredoxin from a Pseudomo-
`nas species, a [2Fe-2S] ferredoxin in the adrenodoxin family (14). This
`sequence information was used to clone the ferredoxin gene by a two-
`step nested PCR approach. Three degenerate oligonucleotide primers
`(two sense and one antisense) were designed and synthesized (Table I).
`The sequence of the two sense primers was based on the N-terminal
`amino acid sequence from the purified ferredoxinDIC. Primer A (17-mer,
`384 variants) was based on the sequence ITVVNQ and primer B (17-
`mer, 192 variants) was derived from the sequence MEVIRD. The se-
`quence of the antisense primer C (17-mer, 8192 variants) was based on
`the amino acid sequence RL(T/S/C)CQ(V/I/L) that was part of the con-
`served [2Fe-2S] domain near the C-terminal end of six previously se-
`quenced bacterial adrenodoxin-type ferredoxins (see Fig. 2). Primers A
`and C were used in a PCR with P. maltophilia genomic DNA as tem-
`plate. An aliquot containing a mixture of the products from the first
`PCR was then used as the template in a second round of amplification
`with primers B and C. A 191-bp product was amplified in the second
`PCR and sequenced. The amino acid sequence predicted by this clone
`matched the N-terminal and internal sequence obtained from the pu-
`rified ferredoxinDIC protein. New sense and antisense primers based on
`the DNA sequence of the clone were designed (Table I) and a 149-bp
`probe was labeled in a PCR. The DIG-labeled probe was hybridized at
`68 °C to P. maltophilia genomic DNA that had been digested with
`several restriction enzymes and blotted to a nylon membrane. A map of
`the restriction sites surrounding the ferredoxin gene was constructed
`based on the sizes of various restriction fragments that hybridized to
`the probe. This restriction map suggested that a full-length ferredoxin
`gene was contained on a 1.0-kb XhoI/PstI fragment. A size-fractionated
`genomic library containing 0.5–1.5-kb XhoI/PstI fragments was con-
`structed and colony lifts were prepared. The 149-bp probe was hybrid-
`ized to ⬃200 bacterial clones from the library at 68 °C and one positive
`clone was selected.
`Cloning of the Oxygenase Gene—The first 27 residues of the N-
`terminal amino acid sequence was determined for the purified oxygen-
`aseDIC protein (Table I) as described previously (10). To clone the
`oxygenase gene, a degenerate oligonucleotide (17-mer, 32 variants)
`based on the sequence NAWYVA (Table I) was designed and synthe-
`sized. Genomic DNA from P. maltophilia was digested with several
`restriction enzymes, resolved on a 1% agarose gel, and blotted to a nylon
`membrane. The labeled oligonucleotide mixture was hybridized to the
`DNA on the blot at temperatures ranging from 35 to 60 °C. At 45 °C, the
`probe detected a single 3.5-kb XhoI/SstII fragment. A size-fractionated
`genomic library containing 3.0 – 4.0-kb XhoI/SstII fragments was con-
`structed and colony lifts were prepared. The oligo probe was hybridized
`to ⬃200 bacterial clones from the library at 45 °C and one positive clone
`was selected.
`Pulsed Field Gel Electrophoresis—A purified plasmid preparation
`from P. maltophilia was resolved on a 0.7% agarose gel in 1⫻ TAE
`buffer by pulsed field gel electrophoresis using a CHEF-DR III system
`(Bio-Rad). The apparatus was run at 6 V for 10 h with an initial switch
`
`time of 2 s and a final switch time of 2 s. The gel was stained with
`ethidium bromide (1 ␮g/ml water) to visualize the plasmid DNA and
`then blotted to a nylon filter. Sizes of megaplasmid DNAs were esti-
`mated relative to two sets of linear DNA markers.
`Expression and Purification of Recombinant Proteins—A set of sense
`and antisense primers was designed for each of the cloned genes to
`introduce an NcoI restriction site at the 5⬘ end and an XhoI restriction
`site at the 3⬘ end of the coding sequence. Each primer pair was used in
`a PCR with Pfu polymerase and the appropriate genomic clone as
`template. The amplified products were digested with NcoI and XhoI and
`then ligated into a pET expression vector (Novagen) that had been
`digested with the same restriction enzymes. The ferredoxin (ddmB) and
`the reductase (ddmA1) genes were cloned into the pET 30b(⫹) vector
`and the oxygenase gene (ddmC) was cloned into the pET 32b(⫹) vector.
`The constructs were sequenced to verify that the coding sequence of
`each gene was in-frame with the vector sequence that encodes a N-
`terminal His6 tag and then transformed into E. coli BL21(DE3) cells
`(Novagen). Recombinant proteins were expressed according to protocols
`in the pET system manual (Novagen). Cells transformed with the
`ferredoxin and reductase constructs were grown in 500 ml of LB me-
`dium supplemented with kanamycin (50 ␮g/ml) at 37 °C with shaking,
`induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside when the
`A600 reached 0.6, and then grown for an additional 3 h at 37 °C.Cells
`transformed with the oxygenase construct were grown in 500 ml of LB
`medium supplemented with ampicillin (75 ␮g/ml) at 37 °C with shak-
`ing, induced with 1 mM isopropyl 1-thio-␤-D-galactopyranoside when
`the A600 reached 0.6, and then grown at 15 °C for an additional 24 h.
`The recombinant proteins were purified on a nickel ion-charged affinity
`column (Novagen) according to the manufacturer’s directions. Fractions
`from the column were analyzed by SDS-PAGE and Coomassie Blue
`staining.
`Assay of Dicamba O-Demethylase Activity—Fractions containing the
`purified native or recombinant reductase, ferredoxin, and oxygenase
`proteins were combined in a standard reaction mixture (9) that was
`assayed for enzymatic activity by using high performance liquid chro-
`matography (HPLC) to monitor the appearance of the DCSA reaction
`product (10). Reactions were performed at 30 °C for 10 min in a total
`volume of 500 ␮l. Assays were initiated by addition of dicamba after a
`5-min preincubation at 30 °C. The reaction was terminated by the
`addition of 80 ␮l of 5% sulfuric acid. Samples were centrifuged, filtered,
`and 250 ␮l of each sample was then injected into a C-18 reverse phase
`␮Bondapack 4.6 ⫻ 150-mm column. The product mixture was separated
`using a linear gradient of 60 to 0% methanol in 40 mM Tris acetate (pH
`7.2) using a Waters HPLC unit. DCSA retention time was determined
`to be 14.1 min (established using 250 ␮l of 500 mM DCSA as a stand-
`ard). Set concentrations of DCSA were used as quantitation standards.
`For kinetic studies, DCSA in reaction samples was detected and quan-
`tified by fluorescence emission at 420 nm (excitation wavelength, 310
`nm) after separation from other reaction products by reverse-phase
`HPLC. Enzymatic activity in fractions containing the particular com-
`ponent being purified was assayed in the presence of excess quantities
`of the other two components of dicamba O-demethylase, 25 mM potas-
`sium phosphate buffer (pH 7.2), 0.5 mM NADH, 10 mM magnesium
`chloride, 0.5 mM ferrous sulfate, and 0.5 mM dicamba.
`
`RESULTS
`Cloning of the Reductase Genes—A 148-bp reductase probe
`was generated by a two-step nested PCR approach described
`under “Experimental Procedures” and used to screen two
`E. coli size-fractionated genomic libraries of P. maltophilia,
`strain DI-6. Two genes (ddmA1 and ddmA2), both of which
`encode the reductase component of dicamba O-demethylase,
`were identified. Sequence analysis showed that a 4.3-kb KpnI/
`EcoRI fragment that hybridized to the probe contained a
`1224-bp open reading frame (ORF) preceded by Shine-
`Dalgarno (ribosome binding site) sequence GGGAAAA posi-
`tioned 11 bases upstream from the initiation codon (data not
`shown). The ORF encoded a 43.7-kDa protein consisting of 408
`amino acids (Fig. 1), a size that was consistent with the
`molecular mass of 45 kDa that was previously estimated for
`purified reductaseDIC by SDS-PAGE (10). The amino acid se-
`quence specified by the ddmA1 gene matched the N-terminal
`and internal amino acid sequence information previously ob-
`tained from purified reductaseDIC. The protein also had a flavin
`binding domain for FAD (consensus sequence TX6AXGD) and
`
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`24762
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`Dicamba O-Demethylase Genes
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`FIG. 1. Alignment of the two predicted amino acid sequences encoding the reductase component of dicamba O-demethylase with
`the sequences of other members of the FAD-dependent pyridine nucleotide reductase family. Proteins in this family have two ADP
`binding domains (for FAD and NADH, respectively) with the consensus sequence GXGX2GX3A and a flavin binding domain (for FAD) with the
`consensus sequence TX6AXGD. DdmA1 (AY786444) and DdmA2 (AY786445), reductase components of dicamba O-demethylase from P. malto-
`philia, strain DI-6; RedA2 (CAA05635), reductase component of dioxin dioxygenase from Sphingomonas sp. RW1; ThcD (P43494), rhodocoxin
`reductase from R. erythropolis; CamA (P16640), putidaredoxin reductase from P. putida (GenBank accession numbers in parentheses).
`
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`Dicamba O-Demethylase Genes
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`24763
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`FIG. 2. Alignment of the predicted amino acid sequence encoding the ferredoxin component of dicamba O-demethylase with the
`sequences of other members of the adrenodoxin family. Proteins in this family contain a [2Fe-2S] domain with the consensus sequence
`CX5CX2CX36/37C. DdmB (AY786442), ferredoxin component of dicamba O-demethylase from P. maltophilia, strain DI-6; CadC (BAB78524),
`ferredoxin component of 2,4-D oxygenase from Bradyrhizobium sp. HW13; FdxP (P37098), ferredoxin from C. crescentus; FdxE (CAA72162),
`ferredoxin from R. capsulatus; ThcC (P43493), rhodocoxin from R. erythropolis (GenBank accession numbers in parentheses).
`
`two ADP binding domains (for FAD and NADH, respectively)
`with the consensus sequence GXGX2GX3A. These conserved
`features were consistent with the yellow color and UV fluores-
`cence previously observed for reductaseDIC (10). The derived
`amino acid sequence was homologous over its entire length to
`other members of the FAD-dependent pyridine nucleotide re-
`ductase family. The identities ranged from 69% with the cyto-
`chrome P450-type reductase component of dioxin dioxygenase
`(RedA2) from Sphingomonas sp. RW1 to 38% with rhodocoxin
`reductase (ThcD)
`from Rhodococcus erythropolis and
`putidaredoxin reductase (CamA) from Pseudomonas putida
`(Fig. 1).
`Sequence analysis showed that a 3.0-kb ApaI fragment that
`hybridized to the same 148-bp reductase probe contained an
`ORF of 1227 bp preceded by a ribosome binding site with the
`sequence GGAG situated 9 bases upstream from the initiation
`codon (data not shown). The coding sequence specified a 43.9-
`kDa protein consisting of 409 amino acids (Fig. 1). The amino
`acid sequence predicted by the second reductase gene (ddmA2)
`was 99.3% identical to the sequence predicted by the first
`reductase gene (ddmA1). As expected, in vitro dicamba O-
`demethylase assays in which DdmA2 was substituted for
`DdmA1 demonstrated that the two enzymes possessed identi-
`cal or nearly identical activities (data not shown).
`Cloning of the Ferredoxin Gene—A 149-bp ferredoxin probe
`was generated by a two-step nested PCR approach described
`under “Experimental Procedures” and used to screen an E. coli
`size-fractionated genomic library of P. maltophilia, strain DI-6.
`A single gene (ddmB) that encodes the ferredoxin component of
`dicamba O-demethylase was identified. Sequence analysis
`showed that a 900-bp XhoI/PstI fragment that hybridized to
`the probe contained an ORF of 315 bp preceded by a ribosome
`binding site with the sequence AGGGGA situated 10 bases
`upstream from the initiation codon (data not shown). The cod-
`ing sequence specified an 11.4-kDa protein composed of 105
`amino acid residues (Fig. 2), a size that was consistent with the
`molecular mass of 14 kDa that was previously estimated for
`purified ferredoxinDIC by SDS-PAGE (10). The amino acid se-
`quence predicted by the ddmB gene matched the N-terminal
`
`and internal amino acid sequence information previously ob-
`tained from purified ferredoxinDIC. The protein also had a
`[2Fe-2S] domain with the consensus sequence CX5CX2-
`CX36/37C, a conserved feature that was consistent with the
`previous EPR spectroscopic analysis of ferredoxinDIC (10). The
`derived amino acid sequence was homologous over its entire
`length to other members of the adrenodoxin family of [2Fe-2S]
`bacterial ferredoxins. The identities ranged from 53% with the
`ferredoxin component of 2,4-D oxygenase (CadC) from Brady-
`rhizobium sp. strain HW13 to 38% with a ferredoxin (FdxP)
`from Caulobacter crescentus (Fig. 2).
`Cloning of the Oxygenase Gene—A 17-mer degenerate oligo-
`nucleotide probe based on the N-terminal amino acid sequence
`of purified oxygenaseDIC was used to screen an E. coli size-
`fractionated genomic library of P. maltophilia, strain DI-6. A
`gene, designated ddmC, which encodes the oxygenase compo-
`nent of dicamba O-demethylase was identified. Sequence anal-
`ysis showed that a 3.5-kb XhoI/SstII fragment that hybridized
`to the probe contained an ORF of 1017 bp preceded by a
`ribosome binding site with the sequence AAGGAG located 7
`bases upstream from the initiation codon (data not shown). The
`coding sequence specified a 37.3-kDa protein composed of 339
`amino acid residues (Fig. 3), a size that was consistent with the
`molecular mass of 40 kDa that was previously estimated for
`purified oxygenaseDIC by SDS-PAGE (10). The amino acid
`sequence predicted by the ddmC gene matched the N-
`terminal sequence information previously obtained from
`purified oxygenaseDIC. In addition, the protein had a Rieske
`[2Fe-2S] domain with the consensus sequence CXHX16CX2H
`and a non-heme Fe(II) domain with the consensus sequence
`(D/E)X3DX2HX4H. Both of these conserved features were con-
`sistent with the previous biochemical characterization of
`oxygenaseDIC (10). The derived amino acid sequence was
`homologous over its entire length to those members of the
`diverse Phthalate family of Rieske non-heme iron oxygenases
`that function as a monooxygenase (11). The identities ranged
`from 36% with the oxygenase component of toluenesulfonate
`methyl-monooxygenase (TsaM) from Comamonas testosteroni
`T-2 to 34% with the oxygenase component of vanillate
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`PGR2023-00022 Page 00005
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`24764
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`Dicamba O-Demethylase Genes
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`FIG. 3. Alignment of the predicted amino acid sequence encoding the oxygenase component of dicamba O-demethylase with the
`sequences of other monooxygenases that are members of the Phthalate family of Rieske non-heme iron oxygenases. Proteins in this
`family are homomultimers containing a Rieske [2Fe-2S] domain with the consensus sequence CXHX16CX2H and a non-heme Fe(II) domain with
`the consensus sequence (D/E)X3DX2HX4H. VanA_Ac (AAC27107), oxygenase component of vanillate demethylase from Acinetobacter sp. ADP1;
`VanA_Ps (O05616), oxygenase component of vanillate demethylase from Pseudomonas sp. HR199; TsaM (AAC44804), oxygenase component of
`toluenesulfonate methyl-monooxygenase from C. testosteroni T-2; DdmC (AY786443) oxygenase component of dicamba O-demethylase from
`P. maltophilia, strain DI-6 (GenBank accession numbers in parentheses).
`
`demethylase (VanA) from Acinetobacter sp. ADP1 (Fig. 3).
`Localization of the Oxygenase Gene (ddmC) to Megaplasmid
`DNA—Plasmid DNA was isolated from cells of P. maltophilia
`that had been grown with dicamba as the