University of Groningen

Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4 Arif, Muhammad Ilan; Samin, Ghufrana; van Leeuwen, Jan G. E.; Oppentocht, Jantina; Janssen, Dick Published in: Applied and Environmental Microbiology

DOI: 10.1128/AEM.00760-12

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Publication date: 2012

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Citation for published version (APA): Arif, M. I., Samin, G., van Leeuwen, J. G. E., Oppentocht, J., & Janssen, D. B. (2012). Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4. Applied and Environmental Microbiology, 78(17), 6128-6136. DOI: 10.1128/AEM.00760-12

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Download date: 11-02-2018 Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

Muhammad Irfan Arif, Ghufrana Samin,* Jan G. E. van Leeuwen , Jantien Oppentocht, and Dick B. Janssen Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, Netherlands

A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloal- Downloaded from cohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. The dppA gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG from Pseudomonas putida HK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of ؊1 1 nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited a kcat of 17 s . H nuclear magnetic resonance experi- ments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferri- http://aem.asm.org/ cyanide [K 3Fe(CN)6] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid.

ichloropropanols are widely used in the chemical industry, nicotinamide . They catalyze the intramolec- Dparticularly as intermediates for epichlorohydrin produc- ular displacement of a halogen by the vicinal hydroxyl group, tion. The classical epichlorohydrin manufacturing process pro- yielding an epoxide, a halide ion, and a proton ( 52 ). At least six ceeds via hydrochlorination of allylchloride, which yields both different halohydrin dehalogenases have been found so far: two 2,3-dichloro-1-propanol and 1,3-dichloro-2-propanol (30 ). Be- from Corynebacterium sp. strain N-1074 (HheA and

cause of the increasing availability of glycerol as a side product HheB) (57 ) and homologs in Arthrobacter sp. strain AD2 on September 27, 2012 by University of Groningen from biodiesel synthesis, this classical process is being replaced (HheBAD2) ( 52 ), Agrobacterium sp. strain NHG3 (DehB) (20 ), Ar- with the use of glycerol as a renewable feedstock for epichlorohy- throbacter erithii H10a (DehA) (5), and Agrobacterium radiobacter drin manufacture, again via chlorination to the same dichloropro- strain AD1 (HheC) (51 ). The structures are known, and the cata- panols or via 1-chloro-2,3-propanediol ( 6). Epichlorohydrin itself lytic mechanism is well understood ( 11 , 12 , 21 ). The halohydrin as well as the production intermediates 2,3-dichloropropanol dehalogenases have a preference for substrates with the halogen (DCP) and 1,3-dichloropropanol are mutagenic, genotoxic, and group on a terminal (primary) carbon atom, which can be ex- carcinogenic, and therefore their release and the possibility of hu- plained by analysis of X-ray structures ( 11 , 21 ). However, impor- man exposure are of significant concern ( 38 ). DCP also occurs as tant compounds, such as 2-chloro-1-propanol and DCP, are not a contaminant in cellulose and starch hydrolysates, soy sauce, and easily converted. The potential importance of DCP as an interme- baked foods (27 ). diate in the degradation of 1,2,3-trichloropropane prompted us to Microorganisms that metabolize dichloropropanols are of in- search for new pathways of DCP metabolism ( 9). terest in view of their role in the removal of these compounds from A well-established mechanism for the conversion of alcohols is waste streams and contaminated environments (14 ), from food oxidative conversion by alcohol dehydrogenases (ADHs). Many and pulp products, and also carbohydrate hydrolysates (55 ) and ADHs are NAD- or NADP-dependent enzymes ( 34 ). Oxidation of because dichloropropanols occur as intermediates in a catabolic alcohols by oxidases, which generates hydrogen peroxide, is also pathway for degradation of the emerging priority contaminant possible. A special class of alcohol dehydrogenases is formed by 1,2,3-trichloropropane (8). Of the dichloropropanols, 2,3-di- the periplasmic quinoprotein ADHs, which contain a quinoid co- chloro-1-propanol is chemically more stable and more difficult to factor, such as pyrroloquinoline quinone (PQQ), and Ca 2ϩ. A degrade than 1,3-dichloropropanol (14 ). Furthermore, microor- quinoprotein methanol dehydrogenase has been found to be re- ganisms that convert dichloropropanols can be used in the prep- sponsible for 2-chloroethanol oxidation in the 1,2-dichloroethane aration of enantiopure building blocks for the pharmaceutical in- catabolic pathway (26 ). PQQ-dependent ADHs have been discov- dustry (27 , 30 ). Several bacterial strains are known to grow on ered in a wide variety of , including Acetobacter, Glucono- dichloropropanols, such as Pseudomonas sp. strain OS-K-29 (30 ), Alcaligenes sp. strain DS-K-S38 (29 ), Mycobacterium sp. strain GP1 (40 ), Agrobacterium sp. strain NHG3 (14 , 20 ), and Arthrobac- Received 7 March 2012 Accepted 14 June 2012 ter sp. strain AD2 (51 ). Published ahead of print 29 June 2012 During the microbial conversion of vicinal haloalcohols, deha- Address correspondence to Dick B. Janssen, [email protected]. logenation is usually the first step, and this reaction can be cata- * Permanent address: Ghufrana Samin, Department of Chemistry, University of lyzed by haloalcohol dehalogenases ( 24 ). These enzymes, also Engineering and Technology Lahore, Faisalabad Campus, Faisalabad, Pakistan. called halohydrin dehalogenases, are composed of 2 to 4 subunits Supplemental material for this article may be found at http://aem.asm.org/. of molecular masses between 28 and 35 kDa and are phylogeneti- Copyright © 2012, American Society for Microbiology. All Rights Reserved. cally related to the short-chain dehydrogenase reductase super- doi:10.1128/AEM.00760-12 family (SDR proteins) (54 ), even though they do not possess a

6128 aem.asm.org Applied and Environmental Microbiology p. 6128–6136 September 2012 Volume 78 Number 17 Quinohemoprotein Dehydrogenase/Dehalogenase

bacter, Pseudomonas, and Comamonas strains (1, 2, 4, 19 , 47 ). characterization. For molecular weight determination of na- Some of these enzymes contain heme as a secondary prosthetic tive enzyme, purified dehydrogenase (DppA) was analyzed by gel filtra- group and are known as quinohemoproteins ( 4). The periplasmic tion (see the supplemental material). The purity and molecular weight of quinohemoproteins transfer electrons to the membrane-bound the protein were determined by SDS-PAGE (12% gel) analysis. Heme bacterial respiratory chain (4, 34 ). staining of the SDS-PAGE gels was done by using the method of Francis In this paper we show that such a quinohemoprotein alcohol and Becker (15 ). To determine heme c concentrations, we measured difference spectra dehydrogenase may act as DCP dehalogenase. We started with the of reduced and oxidized pyridine hemochrome according to a published isolation of a DCP-degrading organism from a site polluted with protocol (53a). chlorinated compounds. We report the properties of this new Enzyme assays. All enzyme assays were performed at 25°C. The dehy-

DCP-utilizing bacterium, analyze the gene encoding the quinohe- drogenase (DppA) activity toward DCP in cell extracts was measured by Downloaded from moprotein alcohol dehydrogenase, and propose a pathway for following the reduction of the electron acceptor DCPIP with PMS as an DCP metabolism. intermediate electron carrier. The reaction mixture (1 ml) contained 50 mM potassium phosphate, pH 7.4, 35 ␮M 2,6-DCPIP, 5 mM DCP, and MATERIALS AND METHODS cell extract. After addition of 1.6 mM PMS, the absorbance was monitored at 600 nm. The absorption coefficient of DCPIP at 600 nm is 21.0 mM Ϫ1 Chemicals, reagents and enzymes. All chemicals were obtained from Alfa cm Ϫ1 (16 ). Aesar, Sigma-Aldrich, and Acros Organics. Oxidase test discs were ob- For routine measurements, the enzyme activity was measured by fol- tained from Fluka. Plasmid DNA was isolated with the Qiagen plasmid lowing the reduction of potassium ferricyanide, K 3Fe(CN)6, at 420 nm in isolation kit. Enzymes used for cloning were either from Roche or New http://aem.asm.org/ a 1-ml reaction mixture consisting of 50 mM Tris-SO , pH 8.0, 5 mM England BioLabs. The PCR master mix for screening was purchased from 4 DCP, and 1 mM potassium ferricyanide (extinction coefficient, 1 mM Ϫ1 Promega. cm Ϫ1 at 420 nm [ 22 ]). One unit of enzyme activity was defined as the Isolation and characterization of strain MC4. The organism used in amount of enzyme catalyzing the reduction of 1 ␮mol of potassium fer- this work, Pseudomonas putida strain MC4, was isolated from mixed sam- ricyanide per min under the conditions used. The kinetic constants were ples of soil collected from a site polluted with chlorinated hydrocarbons obtained by fitting initial rates measured at various substrate concentra- (close to Chemiehaven, Botlek, Rotterdam, Netherlands), using enrich- tions to the Michaelis-Menten equation. ment cultivation with 2 mM DCP as a sole carbon and energy source. Its DCP conversion and product identification. For measuring DCP growth spectra with different halogenated and nonhalogenated com- conversion, chloride production, and product formation, a 50-ml reac-

pounds were determined by replica plating on minimal medium (MMY) on September 27, 2012 by University of Groningen tion mixture was prepared that contained 5 mM DCP as the substrate, 10 agar plates supplemented with the carbon source of choice ( 25 ). The or- mM K Fe(CN) as artificial electron acceptor, and a suitable amount of ganism is deposited at DSMZ under accession number 25823. 3 6 A segment of 16S rRNA was amplified from the genomic DNA of enzyme in 50 mM potassium phosphate buffer, pH 8.0. At several time ␮ strain MC4 by PCR with the universal primers 27F and 1492R ( 33 ), cloned points, a 3-ml sample was withdrawn and quenched with 10 l of 5 M in pZero-2, and sequenced. phosphoric acid. Samples were extracted with diethyl ether (1 ml) con- Growth and enzyme purification. Strain MC4 was grown in a 2.5-liter taining mesitylene as internal standard, and extracts were analyzed on a fermentor in MMY medium containing 5 mM DCP as the sole carbon gas chromatograph containing an HP1 column (30 by 0.25 mm; 0.25 ␮m) source. The inoculum was prepared by growing strain MC4 overnight in according to the following method: 50°C for 5 min, with temperature LB at 30°C. After batch cultivation, cells were collected by centrifugation increase from 50°C to 200°C over 20 min. The carrier gas was helium. and washed in MMY medium. This mixture was added to the batch cul- For chloride measurements, the remaining aqueous layers from di- ethyl ether extractions were analyzed on an ion chromatograph (DX 120; ture to an initial optical density at 600 nm (OD 600) of 0.05. The OD 600 and chloride release were monitored at regular intervals. The pH of the grow- Dionex, Sunnyvale, CA) equipped with an Alltech A-2 anion column (100 ing culture was maintained at 7.0 with 2 M NaOH, and the temperature by 4.6 mm; 7 ␮m) and an Alltech guard column (50 by 4 mm). A mixture of NaHCO and Na CO (3 mM each), pH 10, in deionized water was was maintained at 30°C. At an OD 600 of 0.45, more substrate was added, to 3 2 3 used as eluent at a flow rate of 1.0 ml/min. a total input of 10 mM. At an OD 600 of 0.7, the cells were collected, For identification of the expected aldehyde product of the dehydroge- centrifuged, and washed with 10 mM Tris-SO 4, pH 8.0. The cell pellet was nase reaction, 1 ml of the reaction mixture underwent derivatization by resuspended in 5 volumes of 10 mM Tris-SO 4, pH 8.0, and stored at Ϫ80°C until further use. Five batches obtained in this manner were joined adding dinitrophenyl hydrazine to 5 mM at pH 3.5 ( 3). Acetaldehyde and and sonicated. The lysate was centrifuged at 23,000 ϫ g for 20 min to propanal were used to standardize the derivatization procedure. The de- remove cell debris. The supernatant was again centrifuged at 160,000 ϫ g rived products were separated on a liquid chromatograph-mass spec- for 2 h to separate the membrane fraction and the cell extract. trometry (LC-MS) system (LCQ Fleet ion trap MS; Thermo Scientific) The cell extract was subjected to ammonium sulfate fractionation. equipped with a C 18 Lichrosorb (Agilent Technologies, Santa Clara, CA) Fractions of 55%, 60%, and 65% precipitation were pooled together and reverse-phase column (150 by 3 mm [inner diameter]; 5 ␮m), an electro- desalted with a desalting column (EconoPac 10DG; Bio-Rad Laborato- spray ionization (ESI) ion source, and a photodiode array detector set at ries). The pooled ammonium sulfate fractions were applied to a 60-ml 365 nm. DEAE-Sepharose column (GE Healthcare) preequilibrated with 10 mM To determine the position of chlorine in the reaction product, we

Tris-SO4, pH 8.0. A salt gradient of 1 M NaCl in 10 mM Tris-SO 4, pH 8.0, studied the conversion of DCP using nuclear magnetic resonance (NMR) was used for elution. Active fractions were pooled and then concentrated spectroscopy by recording one-dimensional (1D) proton NMR spectra at with an Amicon filter (Millipore YM30), and the buffer was replaced with 25°C on a Varian Unity Plus 500-MHz spectrometer. The NMR tube (1 10 mM phosphate buffer, pH 8.0. The concentrated fraction was further ml) contained 5 mM DCP, 20 mM potassium ferricyanide, 100 mM po- purified on a ceramic hydroxyapatite (HAP) column preequilibrated with tassium phosphate buffer, pH 8.0, and 20 ␮l of enzyme solution, all in 10 mM potassium phosphate buffer, pH 8.0. The enzyme was eluted with D2O. The reaction was started by adding the enzyme, followed by gentle a gradient of 0.01 to 0.5 M potassium phosphate buffer, pH 8.0, and mixing and recording of 1D 1H NMR spectra for 24 h. Each experiment

concentrated, and the buffer was exchanged with 10 mM Tris-SO 4, pH was performed with 176 scans per transient reaction, an evolution time of 8.0. Fractions of high purity were pooled and stored at Ϫ20°C for further 2 s, and an interscan delay of 3 s, giving rise to a net acquisition time of work. ϳ15 min per spectrum.

September 2012 Volume 78 Number 17 aem.asm.org 6129 Arif et al.

N-terminal sequencing and primer design. The purified DppA pro- The complete gene sequence of the DNA fragment has been deposited tein was subjected to N-terminal sequencing by automated Edman deg- with GenBank under accession number JN162364. The 16S rRNA seg- radation (Eurosequence B.V., Groningen, Netherlands). The resulting se- ment sequence was deposited with GenBank under accession number quence was used in a BLAST search with the NCBI database ( http://blast JF825523. .ncbi.nlm.nih.gov/) to identify homologous sequences. The primers prF1 and prR1, which are based on the N terminus of the protein and a con- RESULTS served region of the homologous quinohemoprotein alcohol dehydroge- Isolation and characterization of strain MC4. Strain MC4 was nases, were used for PCR amplification. The amplified DNA was cloned in pZero-2 and sequenced. Next, the specific primers prF1 and prF2, based isolated from contaminated soil by using enrichment cultivation on the amplified sequence, were used for screening the gene libraries in with DCP as sole carbon and energy source. A growth curve and pLAFR3 and pZero-2 vectors. Another primer, prR3, was used in con- halide assays revealed that all chloride was liberated during DCP Downloaded from junction with the M13FP primer (pZero-2) for screening a second subli- degradation (see Fig. S1 in the supplemental material). Cells of brary in pZero-2, obtained by HindIII digestion. Primer sequences are strain MC4 were Gram negative, motile, and rod shaped. The provided in the supplemental material. organism was oxidase positive and catalase positive and it hydro- Cloning and sequencing of the dichloropropanol dehalogenase gene lyzed starch. Strain MC4 was able to grow on sugars (fructose, region. General procedures for cloning and DNA isolation and manipu- glucose, galactose, and ribose), citrate, acetate, succinate, benzo- lation were performed according to published protocols ( 42 ). ate, primary alcohols, and haloalcohols [3-bromo-2-methyl-1- The genomic DNA of strain MC4 strain was partially digested with propanol, (R)-3-chloro-1,2-propanediol, 3-chloro-1-propanol, Sau3A, and fragments of appropriate size (15 to 30 kb) were cloned into rac-2-chloro-1-propanol, 2-chloroallyl alcohol, and rac-2,3-di- pLAFR3 and packaged (Promega) (44 ). Escherichia coli VCS257 cells were http://aem.asm.org/ chloro-1-propanol], while it did not utilize n-alkanes or 1-chloro- transduced with the packaged mixture, and colonies were selected on LB plates containing 12.5 ␮g/ml tetracycline. Next, colonies were arrayed in n-alkanes as growth substrates. Some haloacids, like 2-bro- microtiter plates containing 100 ␮l of LB medium and incubated for 24 h. moacrylic acid, 2-chloroacrylic acid, rac-2-chloropropionic acid, A PCR-based method ( 37 ) was used for screening the library, using DNA rac-2,3-dichloropropionic acid, and 4-chlorobutyric acid, were extracted from pooled clones. Positive microtiter plate pools were further also good growth substrates for strain MC4. Growth on DCP was screened by columns and rows. The identified positive clones were stored not fast (ca. 0.02 hϪ1); with a 10% inoculum it took 3 days to as glycerol stocks at Ϫ80°C. consume 5 mM DCP in a fermentor (30°C). EcoRI and HindIII were used to generate sublibraries in pZero-2. Li- The 16S rRNA gene sequence fragment of strain MC4 had 99% gation mixtures were transformed into E. coli TOP10 cells (Invitrogen) identity to the rRNA gene of Pseudomonas putida strains ATCC on September 27, 2012 by University of Groningen and selected with 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside 17522, ATCC 17527, and ATCC 17536 (accession numbers and kanamycin (25 ␮g/ml) on LB plates. White colonies were screened by AF094742.1, AF094743.1, and AF094747.1, respectively). This PCR as mentioned above. This yielded a positive EcoRI subclone with a classifies the organism as a strain of P. putida . 2.5-kb insert that was sequenced and found to contain an incomplete Identification and purification of 2,3-dichloropropanol de- sequence of the target gene. From a HindIII sublibrary, another part of the dppA gene was sequenced. The complete gene sequence, including the halogenase. For the conversion of vic-haloalcohols, several halo- putative ribosome binding site and promoter sequence, was assembled hydrin dehalogenases of the SDR superfamily of proteins have and analyzed by using BLAST tools of the NCBI database ( http://blast been described in the literature ( 54 , 57 ). Therefore, we analyzed .ncbi.nlm.nih.gov/). genomic DNA of strain MC4 for the presence of open reading Heterologous expression of DppA in E. coli . The deduced amino acid frames similar to the respective genes for these enzymes. A series of sequence of DppA was analyzed for possible subcellular localization and PCR analyses with the primers derived from the sequences of the N-terminal cleavage sites by using PSORTb v.2.0 ( 17 ) and SignalP v.3.0 hheA, hheB, and hheC genes (54 , 57 ) indicated that no similar gene (7). Based on the predicted peptide cleavage site, a 114-bp forward primer was present in strain MC4. Assays with addition of DCP to cell (prF4) with an NdeI site was designed to replace the 24-amino-acid N- extracts also failed to yield dehalogenase activity, whereas halohy- terminal sequence of DppA with the 21-amino-acid N terminus of E. coli drin dehalogenase activity was readily detected this way in control alkaline phosphatase (accession no. AAA24358). This was used for PCR organisms. with a reverse primer containing a KpnI site (prR4). The PCR product was Next, enzyme activity in cell extracts of strain MC4 grown on cut with NdeI/KpnI and cloned in a pBAD vector to give plasmid pNDL1. Primer sequences are provided in the supplemental material. DCP was tested in a DCPIP reduction assay. The observed reduc- For protein expression, plasmid pNDL1 was cotransformed with plas- tion of DCPIP was dependent on DCP. The specific activity of the mid pEC86, which constitutively produces cytochrome c maturation pro- enzyme in cell extract was 94 mU/mg of protein in the presence of teins (46 ), into E. coli TOP10 (Invitrogen) and JCB712 (23 ). Cells were the artificial electron acceptors DCPIP and PMS, which could not grown in 1 liter of LB medium containing 50 ␮g/ml ampicillin and 175 be replaced by NAD ϩ or NADP ϩ. This suggests that the initial step

␮g/ml chloramphenicol under aerobic conditions at 30°C until the OD 600 in DCP conversion is catalyzed by a dehydrogenase that is sus- reached 0.5. Cultures were induced with 0.02% L-arabinose and incubated pected to simultaneously dechlorinate and transfer electrons to an in a rotary shaker at 17°C and 200 rpm for 24 h. Cells were harvested by acceptor that is not a nicotinamide coenzyme. Activity could also centrifugation, and the periplasmic fractions were obtained by using an be monitored with ferricyanide as an artificial electron acceptor. osmotic shock procedure (17 ). The periplasmic fraction was incubated We called the enzyme DppA. with 100 ␮M PQQ and 1 mM CaCl at 30°C for 30 min to form the 2 The DppA protein was purified in three steps: ammonium sul- holoprotein. Enzyme activity of the recombinant enzyme was measured in the potassium ferricyanide reductase assay mentioned earlier. Heme fate precipitation, ion-exchange chromatography on DEAE-Sep- staining of the periplasmic fraction was also performed to verify incorpo- harose, and separation on a ceramic HAP column ( Table 1; see ration of heme into the active protein. also Fig. S2 in the supplemental material). DCP-dependent ferri- Nucleotide sequence accession numbers. The complete gene se- cyanide reduction was measured at each purification step, in order quence of strain MC4, including the putative ribosome binding site and to identify the protein responsible for the oxidation of the sub- promoter sequence, was determined and the dppA gene was sequenced. strate. The molecular mass of DppA was estimated as 72 kDa by

6130 aem.asm.org Applied and Environmental Microbiology Quinohemoprotein Dehydrogenase/Dehalogenase

TABLE 1 Purification of DppA a (35% identity) (56 ). The first 25 amino acid residues of the en- Protein Total coded DppA protein constitute a typical signal sequence for the concn Activity activity Sp act Recovery Purification translocation of the protein to the periplasmic space. The presence Fraction (mg/ml) (U/ml) (U) (U/mg) (%) factor of a signal peptide is a common characteristic of quinohemopro- CFE 15.3 6.6 265 0.4 100 1 teins (50 ), which are located in the periplasm of Gram-negative (NH4)2SO 4 13.8 18.4 148 1.3 56 3 fractionation bacteria. The predicted size of the mature protein without signal DEAE-Sepharose 0.5 5.8 69 10.9 26 25 Ceramic HAP 0.9 13.2 53 14.1 20 32 peptide is 72,978 kDa, in agreement with the SDS-PAGE analysis. Sequence alignments with proteins of known structure indi- a Enzyme activity was measured at 25°C in 50 mM Tris-SO (pH 8.0) containing 1 mM 4 cated that the amino acids involved in PQQ and calcium binding K3Fe(CN)6 and 5 mM 2,3-dichloropropanol.

in quinohemoproteins and quinoproteins are mostly conserved in Downloaded from DppA (Fig. 2). The PQQ and calcium binding domain, which SDS-PAGE analysis and as 73.5 kDa by gel filtration. This indi- corresponds to the N-terminal part of the sequence, contains sev- cated that the DppA exists as a monomer in its native state. The eral residues that are commonly conserved among quinoproteins enzyme was stable when stored at Ϫ20°C. and quinohemoproteins (e.g., Glu83, Cys129, Cys130, Arg135, Cloning and analysis of the dehalogenase gene. To identify Thr179, Gly195, Glu197, Trp256, Asn274, Trp318, and Asp319), the gene responsible for DCP dehalogenation, the purified protein whereas other residues (Gly194, Ala196, Thr254, Lys346, and was subjected to N-terminal sequencing, which yielded NH 2-QV Trp407) are only conserved in quinohemoproteins. An exception

DQAAIIA, and the NCBI nonredundant protein database was is Gly406 in the PQQ binding domain of DppA, which aligns with http://aem.asm.org/ scanned for homologs. This led to several hits annotated as qui- a conserved Asn present in most quinohemo/quinoproteins, with nohemoprotein dehydrogenases. Multiple sequence alignments the exception of ADH IIG, which has Asp at this position. The showed that these enzymes possess a highly conserved region at acidic residues in the PQQ and calcium domain that are involved about 1 kb downstream of the obtained N-terminal coding se- in catalysis in quinohemo/quinoproteins are conserved as Asp319 quence. Two degenerate primers, prF1 based on the N terminus of and Glu197 in DppA ( 10 ). The heme binding residues Cys616, the protein and prR1 based on the conserved region, were used to Cys619, and His620, which are conserved in the C-terminal heme amplify a segment of the dehalogenase gene. The sequencing of domain of all quinohemoproteins, are also present in DppA, in this fragment confirmed that dppA encoded a quinohemoprotein. agreement with the biochemically observed heme binding. Fi-

To isolate the complete MC4 dehydrogenase/dehalogenase nally, the partially conserved tryptophan docking motifs (W1 to on September 27, 2012 by University of Groningen gene, a gene library of chromosomal DNA of MC4 was con- W8), a typical feature of quinohemo- and quinoproteins ( 4), are structed in pLAFR3. The average size of pLAFR3 cosmids was present in DppA as well. between 45 and 50 kb, based on the restriction analysis of 10 ran- A small open reading frame (ORF) encoding a peptide of 23 dom clones. PCR screening gave six positive clones. One of these amino acids was present downstream of the dppA gene. BLAST was used to generate two separate sublibraries for sequencing, and analysis indicated that this peptide contains glutamate and ty- assembly yielded a 3,555-bp contig containing the complete cod- rosine residues for PQQ biosynthesis (41 ). Upstream of the dppA ing sequence for the DCP dehalogenase gene (dppA) along with its gene, there is an ORF encoding 310 amino acids that constititute a putative ribosome binding site and promoter sequence (Fig. 1). hypothetical protein similar to a putative protein from Azoarcus The complete dppA-encoded protein sequence, including the sp. BH72 (accession number YP_934348) and QbdB from Pseu- signal peptide (698 amino acids [aa]), was very similar to type II domonas spp. (accession number BAC15558). QbdB is a hypo- quinohemoprotein alcohol dehydrogenases, particularly with the thetical protein believed to be involved in the meta-pathway of 2-chloroethanol dehydrogenase from Pseudomonas stutzeri (78% phenol degradation. identity), the homologous alcohol dehydrogenases IIB (76%) and Substrate range and kinetic parameters. Using purified en- IIG (53%) from P. putida HK5 (48 , 49 ), a type I quinohemopro- zyme, the substrate profile of DppA was explored. Table 2 shows tein ethanol dehydrogenase from Comamonas testosteroni (51%) that the enzyme has a broad substrate range. The n-alcohols tested (39 , 45 ), and a tetrahydrofurfuryl alcohol dehydrogenase from were well converted. The diols 1,2-propanediol and 1,3-propane-

Ralstonia eutropha Bo (51% identity) ( 58 ). Homology with less diol gave considerably higher Km values than the other substrates, than 50% identity was found with a quinoprotein ethanol dehy- as also reported for ADH IIG and ADH IIB ( 47 ). The kcat value for Ϫ1 Ϫ1 Ϫ1 drogenase from P. aeruginosa (38% identity) (13 , 31 ) and a meth- DCP was 17.8 s , and the kcat/Km value was 2.3 s ␮M , which anol dehydrogenase from Methylophilus methylotrophus W3A1 indicates that DCP is well converted by DppA.

FIG 1 Schematic overview of the structure of the DCP dehalogenase gene. The direction of transcription is indicated by the large arrows. Small arrows indicate primer positions for amplification. The dppA gene (2.1 kb) encodes the DCP dehalogenase (698 aa). The qbdB gene encodes a putative protein that belongs to a superfamily of proteins involved in the meta-pathway of phenol degradation. The pqqA gene encodes a short protein required for PQQ biosynthesis.

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TABLE 2 Steady-state kinetic parameters and substrate specificities of DppA Ϫ1 Ϫ1 Ϫ1 Substrate kcat (s ) Km (␮M) kcat/K m (s ␮M ) Ethanol 14.9 788 0.02 Propanol 24.6 10 2.5 Butanol 20.2 1.5 13.2 Pentanol 25.3 3.3 7.6 Hexanol 23.9 4.4 5.5 Heptanol 16.1 1.5 10.5 Octanol 14.3 1.1 13.6 Allyl alcohol 17.1 15 1.1 Downloaded from 1,2-Propanediol 7.1 5,613 0.001 1,3-Propanediol 8.5 1,033 0.008 DCP 17.8 7.6 2.3

The purification factor suggests that about 3% of the total pro- tein in cell lysate is DCP dehalogenase which, in combination with FIG 3 Derivatization reaction of chloroacrolein with dinitrophenyl hydra- the kinetic parameters of the dehalogenase and the assumption zine. The presence of the chloroacrolein-dinitrophenylhydrazone adduct ( m/z http://aem.asm.org/ 269.06, M-Hϩ) indicates that chloroacrolein is produced. that about one-fourth of the total cell mass can be recovered as protein in cell extract, suggests a possible DCP degradation rate of ([S] ϫ 0.03 ϫ 0.25 ϫ 14.1)/(0.0076 ϩ [S]) ␮mol/mg of cells/min. This would allow a growth rate of 0.35 h Ϫ1 at 1 mM substrate, indicated that either 2-chloroacrolein or 3-chloroacrolein was assuming that the yield on DCP is the same as on glycerol (ca. 0.06 formed. A DNPH adduct of propanal was also seen in minute mg cell [dry mass]/ ␮mol [59 ]) and no energy generation from the amounts during the initial phase of the enzyme reaction, which dehalogenation reactions. Thus, the observed growth rate of less may have formed via an unidentified side reaction or could hvae Ϫ1 than 0.02 h appears not to be rate limited by the catalytic activity been due to a substrate impurity or fragmentation in the LC-MS. on September 27, 2012 by University of Groningen of the initial dehalogenase. No DNPH adducts indicating the formation of 2-chloropropanal, Product identification. To investigate the mechanism of de- 3-chloropropanal, or 2,3-dichloropropanal were observed. This halogenation, we examined the enzymatic conversion of DCP by indicated that one chlorine was rapidly removed from the sub- purified DppA. Incubation of purified enzyme with substrate and strate during the oxidative reaction. potassium ferricyanide showed that DCP was converted with re- To establish the position of the chlorine released from DCP, lease of chloride, indicating that dechlorination and dehydroge- proton NMR was performed, and chemical shifts were recorded nation take place simultaneously. During conversion of DCP (2.2 for reaction mixtures containing enzyme, DCP, and ferricyanide. mM), 8 mM ferricyanide was reduced (see Fig. S3 in the supple- Three signals in the NMR spectra of an intermediate product were mental material). As homologous quinohemoproteins are alcohol assigned to 2-chloroacrolein ( 43 ). The time course of its aldehyde dehydrogenases that act on the terminal hydroxyl group of alco- proton (9.31 ppm) displayed the same trend as the two alkene hols and diols to form the corresponding aldehydes ( 4), a plausible protons (6.72 and 6.57 ppm). This again suggested that the oxida- mechanism of DCP conversion would be the oxidation to 2,3- tion of the hydroxyl group was accompanied by swift elimination dichloropropanal. This aldehyde could undergo elimination of of HCl, either in the of the enzyme or very rapidly after HCl to form 2- or 3-chloroacrolein. product release from DppA. Two other 1H NMR signals (6.07 and We did not observe an aldehyde product by gas chromatogra- 5.73 ppm) were assigned to the alkene protons of 2-chloroacrylic phy, which could be due to its reactivity or instability, and there- acid, which indicated further oxidation of 2-chloroacrolein to fore we used dinitrophenylhydrazone (DNPH) for derivatization the acid, either by the same purified DppA enzyme or abioti- of the reaction samples and analyzed possible adducts by using cally. The acid apparently was not converted further under LC-MS. In negative ionization mode, different adducts of DNPH these conditions. with aldehydes should give different m/z values, viz m/z 305 The best-separated 1H NMR signals of the starting compound, (DNPH derivative of 2,3-dichloropropanal), m/z 271 (DNPH de- as well as those of the intermediate product and the final product, rivatives of 2- and 3-chloropropanal), or m/z 269 (DNPH deriva- were integrated in all samples and used to visualize substrate con- tive of 2-chloroacrolein and 3-chloroacrolein). The negative- version over time (Fig. 4). The results showed that after 6 h, the mode ESI mass spectra indicated the appearance of a peak at m/z reaction halted at approximately 72% conversion. This was prob- 269 (Fig. 3) that disappeared later during the conversion. This ably due to complete consumption of the electron acceptor. Mass

FIG 2 Sequence alignment of DppA with known quinohemoproteins and quinoproteins. Type II ADHs include 2-chloroethanol dehydrogenase from P. stutzeri (2ClEtDH; accession number AAG09249.1) and the alcohol dehydrogenases ADH IIB (BAC15559.1; PDB 1KV9) and ADH IIG (BAD99293.1; PDB 1YIQ) from P. putida HK5. Type I ADHs include quinohemoprotein ethanol dehydrogenase from C. testosteroni (QH-ADH and Q46444.1; PDB 1KB0) and tetrahydrofur- furyl alcohol dehydrogenase from R. eutropha Bo (THFA-DH; AAF86335.1). Homologous quinoprotein alcohol dehydrogenases are ethanol dehydrogenase from P. aeruginosa (QEDH, CAA08896.1; PDB 1FLG) and methanol dehydrogenase from Methylophylus W3A1 (MEDH, AAA83765.1; PDB 4AAH). Signal sequences are underlined at the beginning of the sequence. Amino acids involved in PQQ and calcium binding are by the letter P on top of the alignment, while those involved in formation of the heme domain are indicated by the letter h. The tryptophan docking motifs W1 to W8 are indicated in boxes.

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FIG 4 Conversion of DCP by DppA, followed by 1H NMR. Symbols: ●, 2,3- DCP; ᮀ, 2-chloroacrolein; ⌬, 2-chloroacrylic acid. FIG 5 Coomassie stain (A) and heme stain (B) of an SDS-PAGE gel contain- ing recombinant DppA expressed in E. coli strains JCB712 (lanes 1 and 2) and balances were not exactly stoichiometric, since only 25% of the TOP10 (lane 3). Different concentrations of arabinose were used for induc- tion: lane 1, 0.002%; lanes 2 and 3, 0.02%. final product was detected based on integration of proton signals. This may be due to the high substrate concentration and the fact that the intermediate 2-chloroacrolein is a very reactive com- http://aem.asm.org/ pound that can form dimers or polymers in aqueous solution ( 43 ), utilization of halogenated organic compounds ( 24 ). The dehalo- especially when produced by a pure enzyme and with few pos- genation of haloalcohols is often catalyzed by haloalcohol dehalo- sibilities for further conversion. Some minor signals in the 1D genases (5, 36 , 54 ), and we initially expected that strain MC4 1H NMR spectra were indeed observed, indicating formation of would also contain such an enzyme, but no such activity could be sideproducts,butthesesignalscouldnotberelatedtoaspecific detected and no homologous dehalogenase gene was present in product. strain MC4. Instead, activity measurements indicated that chlo- Heterologous expression. Since the level of production of the ride release was electron acceptor dependent and led to the iden- native DppA in Pseudomonas strain MC4 was low, further work tification of a novel type of oxidative dehalogenase, which we on September 27, 2012 by University of Groningen aimed at elucidating structure-function relationships in this novel called DppA. The enzyme had a broad substrate range encompass- dehalogenase would benefit from better enzyme production. To ing a number of aliphatic alcohols and aldehydes. We also found facilitate heterologous periplasmic expression in E. coli (18 ), we that DppA accepts both the (R) and ( S) enantiomers of DCP, since fused the dppA gene to the 21-amino-acid signal peptide sequence conversion goes to completion with no sign of biphasic kinetics. of E. coli alkaline phosphatase, yielding the construct pNDL1. As A BLAST search of the sequence of the MC4 dehalogenase gene quinohemoproteins require heme c maturation ( 46 ), the fusion dppA in the NCBI database indicated that DppA was homologous protein was expressed in E. coli JCB712 and E. coli TOP10 in the to type II quinohemoprotein ADHs, which are mostly involved in presence of cytochrome c maturation factors, encoded on plasmid the conversion of nonhalogenated alcohols and contain both pEC86. We found that the recombinantly produced DppA had a PQQ and heme as cofactors ( 4). The native DppA sequence con- higher level of expression and heme incorporation in E. coli tains a 25-aa signal peptide at the N terminus that is cleaved off TOP10(pNDL1)(pEC86) than in E. coli JCB712(pNDL1)(pEC86) during maturation, which was apparent from the N-terminal se- (Fig. 5), even though strain JCB712 is known to incorporate heme quence of the mature isolated protein. Furthermore, dehalogenase effectively in the periplasmic space ( 23 ). The specific activities of activity was detected in the periplasmic fraction prepared from DppA in cell extracts of these recombinant E. coli strains were 1.3 strain MC4 by an osmotic shock method (data not shown). Other U/mg and 0.1 U/mg, respectively, as measured in ferricyanide re- quinohemoproteins also reside in the periplasm ( 50 ). The pres- duction assays. Heme staining confirmed that the DppA protein ence of an enzyme in the periplasmic space may have functional contained covalently bound heme and that the enzyme showed implications, such as improved protein stability and reduced pro- catalytic activity with DCP (Fig. 6). Qualitative analysis indicated teolytic degradation (18 ). Besides, the presence of a dehydroge- that ferricyanide reduction was accompanied by chloride release. nase that forms a reactive and toxic metabolite in the periplasm could suppress potential toxic effects that may occur when forma- DISCUSSION tion of a reactive product occurs in the cytoplasm. The conversion We report the isolation of P. putida strain MC4 from a polluted of 2-chloroethanol by a periplasmic quinoprotein was described site, and this organism is capable of growth on DCP as sole carbon earlier (53 ) and may have the same function: prevention of for- source. The strain grew aerobically on many other compounds as mation of highly reactive chlorinated aldehyde in the cytoplasm. well, including sugars, several halogenated aliphatics, and nonha- Since known quinohemoproteins convert alcohols into corre- logenated alcohols. Bacterial cultures that utilize DCP and 1,3- sponding aldehydes (50 ), we also expected the formation of an dichloropropanol as growth substrates have been described pre- aldehyde during DCP conversion. The results indeed indicated viously, but often the substrate degradation has been incomplete that the first step in the DCP catabolic pathway involves the con- due to enantioselectivity of the catabolic enzymes, which restricts version of DCP into 2-chloroacrolein, which is further converted the possibilities to use such organisms for bioremediation appli- into 2-chloroacrylic acid. Whether the same DppA is solely re- cations (8, 36 ), whereas they may be attractive for production of sponsible for both steps was not certain, but NMR measurements optically active compounds (27 , 28 , 29 , 30 ). indicated that purified DppA was active with 2-chloroacrolein as It is obvious that dehalogenation is a key step for microbial well. By analogy to the well-studied mechanism of quinohemo-

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FIG 6 Proposed mechanism for the two-step oxidation of DCP by DppA. The oxidation results in 2-chloroacrolein and release of chloride. 1, 2,3-dichloro-1-

propanol; 2, 2,3-dichloro-1-propanal; 3, chloroacrolein. http://aem.asm.org/ proteins (50 ), we propose a catalytic mechanism of DppA ( Fig. 6) Purification and characterization of particulate alcohol dehydrogenase that involves dehalogenation in the enzyme’s active site or imme- from Gluconobacter suboxydans. Agric. Biol. Chem. 42 :2045–2056. diately after product release. The 1H NMR experiments indeed 3. Annovazzi L, et al. 2004. High-performance liquid chromatography and capillary electrophoresis: methodological challenges for the determina- suggested that release of chloride and a proton occur immediately tion of biologically relevant low-aliphatic aldehydes in human saliva. Elec- upon formation of 2,3-dichloropropionaldehyde by hydride trophoresis 25 :1255–1263. transfer to PQQ. This forms 2-chloroacrolein, with a structure 4. Anthony C. 2001. Pyrroloquinoline quinone (PQQ) and quinoprotein that is more stable due to resonance delocalization of the ␲-elec- enzymes. Antioxid. Redox Signal. 3:757–774. on September 27, 2012 by University of Groningen trons. Whether the DppA enzyme mechanistically participates in 5. Assis HMS, Sallis PJ, Bull AT, Hardman DJ. 1998. Biochemical charac- terization of a haloalcohol dehalogenase from Arthrobacter erithii H10a. halide release, e.g., through specific stabilizing interactions that Enzyme Microb. Technol. 22 :568–574. facilitate cleavage of the carbon-halogen bond, such as what oc- 6. Bell BM, et al. 2008. Glycerin as a renewable feedstock for epichlorohy- curs with haloalkane and halohydrin dehalogenases (11 , 12 ), is drin production: the GTE process. Clean Soil Air Water 36 :657–661. uncertain at this moment. 7. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. 2004. Improved A somewhat similar oxidative dehalogenation mechanism has prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783–795. 8. Bosma T, Kruizinga E, de Bruin EJ, Poelarends GJ, Janssen DB. 1999. been reported for a flavoenzyme from Alcaligenes sp. DS-S-7G, Utilization of trihalogenated propanes by Agrobacterium radiobacter AD1 and this enzyme has been termed HDDase (28 ). The enzyme ox- through heterologous expression of the haloalkane dehalogenase from idatively dechlorinates (R)-3-chloro-1,2-propanediol and pro- Rhodococcus sp. strain m15-3. Appl. Environ. Microbiol. 65 :4575–4581. duces acetic acid and formic acid. It was suggested that this con- 9. Bosma T, Damborský J, Stucki G, Janssen DB. 2002. Biodegradation of version starts with formation of 3-chloro-2-oxopropanol, which 1,2,3-trichloropropane through directed evolution and heterologous ex- pression of a haloalkane dehalogenase gene. Appl. Environ. Microbiol. could be cleaved by the reductive action of the FADH 2-containing 68 :3582–3587. enzyme (27 ). The DppA-catalyzed dehalogenation is mechanisti- 10. Chen ZW, et al. 2002. Structure at 1.9 Å resolution of a quinohemopro- cally completely different from the halohydrin dehalogenase-cat- tein alcohol dehydrogenase from Pseudomonas putida HK5. Structure 10 : alyzed dehalogenation of chloroalcohols, where the vicinal halo- 837–849. 11. de Jong RM, et al. 2003. Structure and mechanism of a bacterial haloal- gen is released and an epoxide is formed by an intramolecular cohol dehalogenase: a new variation of the short-chain dehydrogenase/ nucleophilic substitution (11 ). reductase fold without an NAD(P)H binding site. EMBO J. 22 :4933–4944. Further degradation of 2-chloroacrylic acid was not studied 12. de Jong RM, Dijskstra BW. 2003. Structure and mechanism of bacterial in MC4, but known pathways are hydrolytic dechlorination of dehalogenases: different ways to cleave a carbon-halogen bond. Curr. 2-chloroacrylic acid, which yields pyruvate (32 ), or reduction of Opin. Struct. Biol. 13 :722–730. 13. Diehl A, Wintzingerode FV, Gorisch H. 1998. Quinoprotein ethanol 2-chloroacrylic acid to 2-chloropropionic acid, which can be de- dehydrogenase of Pseudomonas aeruginosa is a homodimer: sequence of halogenated to lactate ( 35 ). the gene and deduced structural properties of the enzyme. Eur. J. Biochem. 257:409–419. ACKNOWLEDGMENTS 14. Effendi AJ, Greenaway SD, Dancer BN. 2000. Isolation and character- This work was supported by EU project EVK1-CT-1999-00023 ization of 2,3-dichloro-1-propanol-degrading rhizobia. Appl. Environ. Microbiol. 66 :2882–2887. (MAROC) and through B-Basic, a public-private NWO-ACTS program. 15. Francis RT, Becker RR. 1984. Specific indication of hemoproteins in G. Samin and M.I. Arif acknowledge support by the Higher Education polyacrylamide gels using a double-staining process. Anal. Biochem. 136: Commission (HEC), Government of Pakistan. 509–514. REFERENCES 16. Furuta S, Mayazawa S, Hashimoto T. 1981. Purification and properties of rat liver acyl-CoA dehydrogenases and electron transfer flavoprotein. J. 1. Adachi O, Miyagawa E, Shinagawa E, Matsushita K, Ameyama M. 1978. Biochem. 90 :1739–1750. Purification and properties of particulate alcohol dehydrogenase from 17. Gardy JL, et al. 2005. PSORTb v. 2.0: expanded prediction of bacterial Acetobacter aceti. Agric. Biol. Chem. 42 :2331–2340. protein subcellular localization and insights gained from comparative 2. Adachi O, Tayama K, Shinagawa E, Matsushita K, Ameyama M. 1978. proteome analysis. Bioinformatics 21 :617–623.

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18. Georgiou G, Segatori L. 2005. Preparative expression of secreted proteins 39. Oubrie A, Rozeboom HJ, Kalk KH, Huizinga EG, Dijkstra BW. 2002. in bacteria: status report and future prospects. Curr. Opin. Biotechnol. Crystal structure of quinohemoprotein alcohol dehydrogenase from Co- 16 :538–545. mamonas testosteroni: structural basis for substrate oxidation and electron 19. Groen BW, van Kleef MAG, Duine JA. 1986. Quinohaemoprotein alco- transfer. J. Biol. Chem. 277:3727–3732. hol dehydrogenase apoenzyme from Pseudomonas testosteroni. Biochem. 40. Poelarends GJ, Hylckama Vlieg JETV, Marchesi JR, Freitas dos Santos J. 234:611–615. LM, Janssen DB. 1999. Degradation of 1,2-dibromoethane by Mycobac- 20. Higgins TP, Hope SJ, Effendi AJ, Dawson S, Dancer BN. 2005. Bio- terium sp. strain GP1. J. Bacteriol. 181:2050–2058. chemical and molecular characterization of the 2,3-dichloro-1-propanol 41. Puehringer S, Metlitzky M, Schwarzenbacher R. 2008. The pyrrolo- dehalogenase and stereospecific haloalkanoic dehalogenases from a versa- quinoline quinone biosynthesis pathway revisited: a structural approach. tile Agrobacterium sp. Biodegradation 16 :485–492. BMC Biochem. 9:8. doi:10.1186/1471-2091-9-8. 21. Hopmann KH, Himo F. 2008. Quantum chemical modeling of the de- 42. Sambrook J, Russell DW. 2001. Molecular cloning: a laboratory manual, halogenation reaction of haloalcohol dehalogenases. J. Chem. Theory 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Downloaded from Comput. 4:1129–1137. 43. Schuphan I, Casida JE. 1979. S-Chloroallyl thiocarbamate herbicides: 22. Ibers JA, Davidson N. 1951. On the interaction between hexacyanofer- chemical and biological formation and rearrangement of diallate and tri- rate (III) ions and (a) hexacyanoferrate (II) or (b) iron (III) ions. J. Am. allate sulfoxides. J. Agric. Food Chem. 27 :1060–1067. Chem. Soc. 73 :476–478. 44. Staskawicz B, Dahlbeck D, Keen N, Napoli C. 1987. Molecular charac- 23. Iobbi-Nivol C, et al. 1994. A reassessment of the range of c-type cyto- terization of cloned avirulence genes from race 0 and race 1 of Pseudomo- chromes synthesized by Escherichia coli K-12. FEMS Microbiol. Lett. 119: nas syringae pv. glycinea. J. Bacteriol. 169:5789–5794. 89–94. 45. Stoorvogel J, et al. 1996. Characterization of the gene encoding quino- 24. Janssen DB. 2007. Biocatalysis by dehalogenating enzymes. Adv. Appl. Microbiol. 61 :233–252. haemoprotein ethanol dehydrogenase of Comamonas testosteroni. Eur. J. Biochem. 235:690–698.

25. Janssen DB, et al. 1989. Cloning of 1,2-dichloroethane degradation genes http://aem.asm.org/ of Xanthobacter autotrophicus GJ10 and expression and sequencing of the 46. Thony-Meyer L. 1997. Biogenesis of respiratory cytochromes in bacteria. dhlA gene. J. Bacteriol. 171:6791–6799. Microbiol. Mol. Biol. Rev. 61 :337–376. 26. Janssen DB, Keuning S, Witholt B. 1987. Involvement of a quinoprotein 47. Toyama H, et al. 1995. Three distinct quinoprotein alcohol dehydroge- alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase nases are expressed when Pseudomonas putida is grown on different alco- in 2-chloroethanol metabolism in Xanthobacter autotrophicus GJ10. J. hols. J. Bacteriol. 177:2442–2450. Gen. Microbiol. 133:85–92. 48. Toyama H, Fujii T, Aoki N, Matsushita K, Adachi O. 2003. Molecular 27. Kasai N, Suzuki T, Idogaki H. 2006. Enzymatic degradation of esters of cloning of quinohemoprotein alcohol dehydrogenase, ADH IIB, from dichloropropanols: removal of chlorinated glycerides from processed Pseudomonas putida HK5. Biosci. Biotoechnol. Biochem. 67 :1397–1400. foods. LWT Food Sci. Technol. 39 :86–90. 49. Toyama H, et al. 2005. Molecular cloning and structural analysis of 28. Kasai N, Suzuki T, Furukawa Y. 1998. Optically active chlorohydrins as quinohemoprotein alcohol dehydrogenase ADH-IIG from Pseudomonas on September 27, 2012 by University of Groningen chiral C3 and C4 building units: microbial resolution and synthetic appli- putida HK5. J. Mol. Biol. 352:91–104. cations. Chirality 10 :682–692. 50. Toyama H, Mathews FS, Adachi O, Matsushita K. 2004. Quinohemo- 29. Kasai N, Tsujimura T, Unoura K, Suzuki T. 1992. Isolation of ( S)-2,3- protein alcohol dehydrogenases: structure, function and physiology. dichloro-1-propanol assimilating bacterium, its characterization, and its Arch. Biochem. Biophys. 428:10–21. use in preparation of ( R)-2,3-dichloro-1-propanol and ( S)- 51. van den Wijngaard AJ, Janssen DB, Witholt B. 1989. Degradation of epichlorohydrin. J. Ind. Microbiol. 10 :37–43. epichlorohydrin and halohydrins by bacteria isolated from freshwater 30. Kasai N, Tsujimura K, Unoura K, Suzuki T. 1990. Degradation of sediment. J. Gen. Microbiol. 135:2199–2208. 2,3-dichloro-1-propanol by a Pseudomonas species. Agric. Biol. Chem. 52. van den Wijngaard AJ, Reuvekamp PTW, Janssen DB. 1991. Purifica- 54 :3185–3190. tion and characterization of haloalcohol dehalogenase from Arthrobacter 31. Keitel T, et al. 2000. X-ray structure of the quinoprotein ethanol dehy- sp. strain AD2. J. Bacteriol. 173:124–129. drogenase from Pseudomonas aeruginosa: basis of substrate specificity. J. 53. van der Ploeg JR, Kingma J, De Vries EJ, Van der Ven JG, Janssen DB. Mol. Biol. 297:961–974. 1996. Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by 32. Kurata A, Kurihara T, Kamachi H, Esaki N. 2005. 2-Haloacrylate re- overexpression of an NAD dependent aldehyde dehydrogenase with low ductase, a novel enzyme of the medium chain dehydrogenase/reductase affinity for halogenated aldehydes. Arch. Microbiol. 165:258–264. superfamily that catalyzes the reduction of a carbon-carbon double bond 53a.Vangnai AS, Arp DJ. 2001. An inducible 1-butanol dehydrogenase, a of unsaturated organohalogen compounds. J. Biol. Chem. 280:20286– quinohemoprotein, is involved in the oxidation of butane by Pseudomonas 20291. butanovora. Microbiology 147:745–756. 33. Lane DJ. 1991. 16S/23S rRNA sequencing, p 115–175. In Stackebrandt E, 54. van Hylckama Vlieg JE, et al. 2001. Halohydrin dehalogenases are struc- Goodfellow M (ed), Nucleic acid techniques in bacterial systematics. John turally and mechanistically related to short-chain dehydrogenases/ Wiley & Sons, Chichester, England. reductases. J. Bacteriol. 183:5058–5066. 34. Matsushita K, Toyama H, Adachi O. 1994. Respiratory chains and bio- 55. Velisek J, Davidek J, Simicova Z, Svobodava Z. 1982. Glycerol chloro- energetics of acetic acid bacteria. Adv. Microb. Physiol. 36 :247–301. 35. Mowafy AM, Kurihara T, Kurata A, Uemura T, Esaki N. 2010. 2-Halo- hydrins and their esters: reaction products of lipids with hydrochloric acrylate hydratase, a new class of flavoenzyme that catalyzes the addition acid. Sci. Pap. Prague Inst. Chem. Technol. E 53 :55–65. of water to the substrate for dehalogenation. Appl. Environ. Microbiol. 56. Xia Z, et al. 1996. Determination of the gene sequence and the three- 76 :6032–6037. dimensional structure at 2.4 angstroms resolution of methanol dehydro- 36. Nakamura T, Nagasawa T, Yu F, Watanabe I, Yamada H. 1992. Reso- genase from Methylophilus W3A1. J. Mol. Biol. 259:480–501. lution and some properties of enzymes involved in enantioselective trans- 57. Yu F, Nakamura T, Mizunashi W, Watanabe I. 1994. Cloning of two formation of 1,3-dichloro-2-propanol to ( R)-3-chloro-1,2-propanediol halohydrin hydrogen-halide- genes of Corynebacterium sp. strain by Corynebacterium sp. strain N-1074. J. Bacteriol. 174:7613–7619. N-1074 and structural comparison of the genes and gene products. Biosci. 37. Ohtsuka M, et al. 2002. Rapid screening of a novel arayed Medaka ( Ory- Biotechnol. Biochem. 58 :1451–1457. zias latipes) cosmid library. Mar. Biotechnol. 4:173–178. 58. Zarnt G, Schrader T, Andreesen JR. 2001. Catalytic and molecular prop- 38. Omura M, Hirata M, Zhao M, Tanaka A, Inoue N. 1995. Comparative erties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase testicular toxicities of two isomers of dichloropropanol, 2,3-dichloro-1- from Ralstonia eutropha strain Bo. J. Bacteriol. 183:1954–1960. propanol, and 1,3-dichloro-2-propanol, and their metabolites alpha- 59. Zhu J, Sánchez A, Bennett GN, San KY. 2011. Manipulating respiratory chlorohydrin and epichlorohydrin, and the potent testicular toxicant 1,2- levels in Escherichia coli for aerobic formation of reduced chemical prod- dibromo-3-chloropropane. Bull. Environ. Contam. Toxicol. 55 :1–7. ucts. Metab. Eng. 13 :704–712.

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