Thermostable Flavin Reductase That Couples with Dibenzothiophene Monooxygenase, from Thermophilic Bacillus Sp

Biosci. Biotechnol. Biochem., 68 (8), 1712–1721, 2004

Thermostable Flavin Reductase That Couples with Dibenzothiophene Monooxygenase, from Thermophilic Bacillus sp. DSM411: Puriﬁcation, Characterization, and Gene Cloning

Takashi OHSHIRO, Hiroko YAMADA, Tomohisa SHIMODA, y Toshiyuki MATSUBARA, and Yoshikazu IZUMI

Department of Biotechnology, Tottori University, Tottori 680-8552, Japan

Received April 5, 2004; Accepted June 1, 2004

Flavin reductase is essential for the oxygenases enzyme from V. fischeri has been determined.13) In involved in microbial dibenzothiophene (DBT) desul- addition, multiple flavin reductases were found in furization. An enzyme of the thermophilic strain, Escherichia coli and Bacillus subtilis, and some of Bacillus sp. DSM411, was selected to couple with DBT them were shown to have nitroreductase activity monooxygenase (DszC) from Rhodococcus erythropolis catalyzing the reduction of aromatic nitrocompounds D-1. The flavin reductase was purified to homogeneity other than flavin compounds.14–16) The crystal structures from Bacillus sp. DSM411, and the native enzyme was a of the enzymes from E. coli have been solved.17,18) monomer of Mr 16 kDa. Although the best substrates We have studied microbial dibenzothiophene (DBT) were flavin mononucleotide and NADH, the enzyme also desulfurization in its enzymological and molecular used other flavin compounds and acted slightly on biological aspects.19,20) The sulfur-specific metabolic nitroaromatic compounds and NADPH. The purified pathway of DBT was investigated using two mesophilic enzyme coupled with DszC and had a ferric reductase Rhodococcus strains, R. erythropolis IGTS8 and activity. Among the flavin reductases so far character- R. erythropolis D-1.19,20) DBT was initially oxidized ized, the present enzyme is the most thermophilic and by two monooxygenases, DBT monooxygenase (DszC) thermostable. The gene coded for a protein of 155 amino and DBT sulfone monooxygenase (DszA), and finally acids with a calculated mass of 17,325 Da. The enzyme converted to 2-hydroxybiphenyl by the desulfinase was overproduced in Escherichia coli, and the specific DszB. We have purified and characterized all these activity in the crude extracts was about 440-fold higher desulfurization enzymes.21–23) Flavin reductase was than that of the wild-type strain, Bacillus sp. DSM411. necessary for the activities of these two monooxygenases involved in the microbial DBT desulfurization.24) Key words: flavin reductase; desulfurization; oxygenase; We have purified, characterized, and overexpressed Bacillus; ferric reductase flavin reductase from the desulfurizing bacterium R. erythropolis D-1.25) Flavin reductase was also puri- Flavin reductase catalyzes the reduction of flavins, fied from the thermophilic desulfurization bacterium such as flavin mononucleotide (FMN), flavin adenine Paenibacillus sp. A11-2,26) and its gene was cloned.27) It dinucleotide (FAD), and riboflavin by NADH and/or has been reported that flavin reductases from V. harveyi NADPH to form reduced flavins. It has been reported and Photobacterium fischeri stimulated the enzyme that reduced flavins are required for a number of activities of various monooxygenases from different monooxygenase enzyme systems. The first example was microorganisms.5,24) We have reported that flavin re- demonstrated in luciferase of the luminous bacterium ductase from the non-desulfurizing bacterium Paeniba- Vibrio harveyi, which catalyzes the oxidation of a long- cillus polymyxa A-1 was more effective than that from chain aliphatic aldehyde by oxygen in the presence of a the desulfurizing bacterium R. erythropolis D-1,28) and reduced flavin.1) Such monooxygenase systems have worked well for DBT desulfurization in recombinant also been found in the biosynthesis of antibiotics, E. coli.29) actinorhodin,2) pristinamycin,3) and valanimycin,4) and Although many flavin reductases were studied as in the degradation of ethylenediamine tetraacetate,5) described above, little information is available about the nitrilotriacetate,6) aliphatic sulfonates,7,8) and 4-hydroxy- thermostability of the enzymes. The microbial desulfur- phenylacetate.9,10) The properties of flavin reductases ization of petroleum, if industrialized in the future, is from luminous bacteria have been studied extensive- postulated to be carried out after the current chemical ly,11,12) and the three-dimensional structure of the desulfurization treatment, which is run at 300 Cor

y To whom correspondence should be addressed. Tel/Fax: +81-857-31-5267; E-mail: [email protected] Flavin Reductase from a Thermophilic Bacterium 1713 higher. Hence, microbial desulfurization at high temper- ing 0.1 M KCl. The active fractions were collected and atures is desirable to minimize cooling costs.30–32) We concentrated by ultrafiltration. searched thermophilic flavin reductases among those Phenyl-Toyopearl. The enzyme solution obtained in that couple with the monooxygenase DszC, from step 1 was dialyzed against the basal buffer containing thermophilic non-desulfurizing bacteria, and selected 2 M (NH4)2SO4. The solution was applied to a Phenyl- the enzyme of Bacillus sp. DSM411. In this study, we Toyopearl 650M column (2:5 18 cm) which had been purified and characterized flavin reductase from this equilibrated with the same buffer. The column was strain, and cloned and expressed its gene in E. coli.We washed with the same buffer and buffer containing 1.6 M report that the enzyme was characteristic in terms of (NH4)2SO4, and bound proteins were eluted with the stability against heat and pH. buffer containing 1.4 M (NH4)2SO4. The active fractions were collected and concentrated by ultrafiltration. Materials and Methods FMN agarose. The enzyme solution obtained in step 2 was dialyzed against 1.5 M potassium phosphate buffer Materials. Q-Sepharose Fast Flow and Superdex (pH 8.0) containing 1 mM DTT and 10% glycerol. The HR10/30 were obtained from Amersham Pharmacia solution was applied to an FMN agarose column (Uppsala, Sweden). Phenyl-Toyopearl 650M was pur- (1 6 cm) which had been equilibrated with the same chased from Tohso (Tokyo, Japan). FMN agarose was buffer. The column was washed with the same buffer purchased from Sigma (St. Louis, Missouri., U.S.A). and 0.9 M potassium phosphate buffer (pH 8.0) contain- Calibration proteins for sodium dodecyl sulfate poly- ing 1 mM DTT and 10% glycerol, and bound proteins acrylamide gel electrophoresis (SDS-PAGE) and for gel were eluted with distilled water containing 1 mM DTT chromatography were obtained from Amersham Phar- and 10% glycerol. The active fractions were collected macia and Roche Diagnostics (Mannheim, Germany) and concentrated by ultrafiltration. respectively. Restriction endonucleases were purchased Superdex HR10/30. The buffer solution dissolving the from Toyobo (Osaka, Japan) or Promega (Madison, enzyme in step 3 was replaced by ultrafiltration with Wisconsin., U.S.A). Three vectors, pGEM-7, pBlue- 50 mM Tris–HCl buffer (pH 8.0) containing 1 mM DTT. script II SK-, and pET-21a were obtained from Promega, The enzyme solution was applied to a Superdex 200 HR Toyobo, and Novagen (Madison) respectively. 10/30 (1 30 cm) equilibrated with the same buffer. The chromatography was performed at a flow rate of Medium and cultivation. Bacillus sp. DSM411 was 0.25 ml/min controlled by an AKTA system (Amersham grown in a nutrient medium containing 5 g of Poly- Pharmacia). The active fractions were concentrated by pepton, 2 g of glycerol, 2 g of yeast extract, 2 g of meat ultrafiltration. extract, 2 g of K2HPO4,1gofKH2PO4, 0.1 g of MgSO4.7H2O, and 0.05 g of CaCl2 in 1000 ml of Enzyme assay. Flavin reductase activity was deter- distilled water (pH 7.2). Cultivation was done in 2-liter mined at 65 C using the decrease in absorbance at flasks containing 500 ml of the medium with reciprocal 340 nm due to oxidation of NADH. The reaction mixture shaking at 130 strokes/min at 55 C for 18 h. The cells contained 20 mM potassium phosphate buffer (pH 7.0), were collected by centrifugation at 8,000 g and stored 0.5 mM NADH, 20 M FMN, and the enzyme in a total at 20 C. Frozen cells (390 g, wet weight) obtained volume of 0.5 ml. One unit of activity was defined as the from 65 liters of the culture broth were thawed and amount of flavin reductase necessary to decrease 1 mol suspended in 50 mM Tris–HCl buffer (pH 8.0) contain- of NADH per min. The coupling assay with DszC of ing 0.5 mM phenylmethanesulfonyl fluoride (PMSF), R. erythropolis D-1 was done at 35 C, and the rate of 1mM dithiothreitol (DTT), and 10% glycerol. This conversion of DBT to DBT sulfone was measured using buffer, except for PMSF, is referred to as the basal the high-performance liquid chromatography system as buffer. Then the cells were disrupted with an ultrasonic described previously.21) The reaction mixture contained oscillator (Sonifier 450; Branson Instruments, Danbury, 100 mM potassium phosphate buffer (pH 7.0), 0.27 mM Connecticut, U.S.A) at 20 kHz. The cell debris was DBT, 6 mM NADH, 10 M FMN, DszC, and the enzyme removed by centrifugation at 14,000 g for 90 min, and in a total volume of 0.25 ml. Purified DszC of the resultant supernatant was used as the cell-free R. erythropolis D-1 was prepared as described previ- extract. ously21) and used in the reaction mixtures. Ferric reductase activity was determined using ferricyanide as Purification of flavin reductase from Bacillus sp. an electron acceptor according to the method previously DSM411. All purification steps were performed at 4 C. reported.33) The reaction mixture contained 50 mM Q-Sepharose Fast Flow. Cell-free extracts were potassium phosphate buffer (pH 7.0), 1 mM NADH, dialyzed against the basal buffer with 0.5 mM PMSF. 5 M FMN, 1 mM potassium hexacyanoferrate (III), and The solution was applied to a Q-Sepharose column the enzyme in a total volume of 0.5 ml. Enzyme activity (5 50 cm) which had been equilibrated with the same was determined spectrophotometrically by monitoring buffer. The column was washed with the same buffer the decrease in absorption of ferricyanide at 420 nm 1 1 and bound proteins were eluted with the buffer contain- ("420 ¼ 1020 M cm ). The control experiments were 1714 T. OHSHIRO et al. done in the absence of FMN or ferricyanide. One unit of 68 Cin0:1 SSC containing 0.1% SDS. Then the activity was defined as the amount of the enzyme membrane was incubated with antidigoxygenin conju- necessary to decrease 1 mol of ferricyanide per min. gated with alkali phosphatase, and the hybridized fragments were detected with 4-nitroblue tetrazolium DNA technique and PCR. Isolation of genomic and chloride and 5-bromo-4-chloro-3-indolyl phosphate. For plasmid DNA, transformation, and restriction endonu- colony hybridization, the gene library of the genomic clease digestion, as well as other recombinant DNA DNA on the agar plate was transferred onto the nylon techniques, were performed as described by Sambrook membrane. The subsequent procedure was the same as et al.34) DNA fragments were purified from an agarose described above. gel by using a Sephaglas BandPrep kit (Amersham Pharmacia). DNA sequencing was performed with Purification of flavin reductase from the recombinant double stranded templates using a DNA sequencing kit E. coli. The recombinant E. coli BL21 (DE3) harboring (Applied Biosystems, Foster City, California, U.S.A) the overexpression vector was cultured in 500 ml flasks based on Taq DNA polymerase-initiated cycle sequenc- containing 100 ml of Luria-Bertani medium supplement- ing reactions with a dye termination kit using a model ed with 100 mg of ampicillin per liter at 37 C. After PRISM 3100 DNA sequencer (Applied Biosystems). cultivation for 2.5 h, 1 mM isopropyl--D-thiogalacto- Primers were designed for cloning the flavin reductase pyranoside was added to the medium, and cultivation gene based on the N-terminal and internal amino acid was continued for an additional 5.5 h. The cells (1.22 g, sequences of the enzyme. PCR was performed with Taq wet weight) from 200 ml of the culture broth were DNA polymerase using the total DNA of Bacillus sp. harvested by centrifugation at 8,000 g for 20 min, and DSM411 as a template under the conditions recom- suspended in the basal buffer with 0.5 mM PMSF and mended by the manufacturer. The reaction mixture was 1mM ethylenediamine tetraacetate (EDTA). The cells heated at 96 C for 5 min, then subjected to 30 cycles of were disrupted by sonication and centrifuged at 95 C for 1 min, 45 C for 1 min, and 72 C for 1 min 10,000 g for 30 min to remove cell debris. with primers FRNshi-5, 50-ATGGACGATCGNACNT- Q-Sepharose Fast Flow. The cell-free extracts pre- TYMG-30, and FRCshi-7, 50-GGATCNCCNGGYTCR- pared were dialyzed against the basal buffer with 0.5 mM 0 TC-3 . For overexpression of the gene, PCR was done PMSF and 1 mM EDTA. The solution was applied to a with an Expand High Fidelity PCR System (Roche Q-Sepharose column (2 11 cm) equilibrated with the Diagnostics) using the plasmid pFRS5 harboring the same buffer. The column was washed with the same structural gene of flavin reductase as a template, and buffer, and bound proteins were eluted with a linear 0 primers, FRBSEC-N (5 -GAACCATATGGATGATC- gradient of 0 to 0.15 M KCl in the buffer. The active GTACATTTCGCAG-30 [the NdeI restriction site is fractions were collected and concentrated by ultrafiltra- underlined and the ATG initiation codon is indicated by tion. The chromatography was repeated twice by using bold type]) and T7-primer (50-GTAATACGACTCAC- NaCl instead of KCl. TATAGGGC-30). The reaction mixture was heated at Ammonium sulfate fractionation. Solid ammonium 96 C for 2 min, then subjected to 30 cycles of 96 C for sulfate was added to the enzyme solution at 2 M. The 1 min, 57 C for 1 min, and 72 C for 1 min. Amplified resultant supernatant was recovered after 24 h by DNA fragments were digested with NdeI and SalI, centrifugation at 10,000 g for 30 min and dialyzed separated by agarose gel electrophoresis, inserted into against the basal buffer. pET-21a vector digested with the same restriction Superdex HR10/30. The enzyme solution was con- enzymes, and then used to transform E. coli JM109 centrated to 5 mg/ml by ultrafiltration, replacing the cells. To overproduce flavin reductase by the pET buffer solution dissolving the enzyme with a 50 mM system, E. coli BL21 (DE3) was transformed with the Tris–HCl buffer (pH 8.0) containing 1 mM DTT, applied constructed plasmid. to a Superdex 200 HR 10/30 (1 30 cm) equilibrated with the same buffer. The chromatography was per- Southern hybridization. Southern hybridization was formed at a flow rate of 0.25 ml/min controlled by an performed using the DIG DNA Labeling kit (Roche AKTA system. The active fractions were concentrated Diagnostics). The probe for cloning of flavin reductase by ultrafiltration. was prepared with digoxigenin (DIG)-11-dUTP by PCR. MonoQ HR10/10. The enzyme solution was applied Total DNA of Bacillus sp. DSM411 was digested with to a MonoQ HR10/10 (1 10 cm) equilibrated with restriction enzymes and separated by agarose gel 50 mM Tris–HCl buffer (pH 8.0) containing 1 mM DTT. electrophoresis. Digested DNA was transferred onto The chromatography was done with a linear gradient of the nylon membrane according to the manufacturer’s 0 to 0.15 M NaCl in the buffer at a flow rate of 0.25 ml/ instructions. After prehybridization, the membrane was ml. The active fractions were concentrated by ultra- hybridized with a DIG-labeled probe at 68 C for 16 h. filtration. The membrane was washed twice at room temperature in 2 SSC (1 SSC is 0.15 M NaCl plus 0.015 M Further methods. The native molecular mass was sodium citrate, pH 7.0) containing 0.1% SDS, and at determined by the AKTA system with a Superdex 200 Flavin Reductase from a Thermophilic Bacterium 1715 Table 1. Purification of Flavin Reductase from Bacillus sp. DSM411

Total protein Total activity Specific activity Purification Yield Purification step (mg) (units) (units/mg) (fold) (%) Cell-free extract 9,400 2,120 0.23 1 100 Q-Sepharose 869 2,150 2.5 10.9 101 Phenyl-Toyopearl 74.5 1,450 19.5 84.8 68.4 FMN agarose 2.1 531 253 1,100 25.0 Superdex 0.6 163 272 1,180 7.7

HR 10/30 column at a ﬂow rate of 0.25 ml/min using 50 mM Tris–HCl (pH 8.0) buﬀer containing 1 mM DTT. The calibration proteins used were aldolase (158 kDa), albumin (45 kDa), chymotrypsinogen A (25 kDa), and cytochrome c (12.5 kDa). Protein concentrations were measured by the method of Bradford35) using bovine serum albumin as the standard. The N-terminal and internal amino acid sequences of the enzyme were determined with a PPSQ-10 protein sequencer (Shimadzu, Kyoto, Japan). The internal amino acid fragment was obtained by CNBr treatment.

Results

Purification of flavin reductase from Bacillus sp. Fig. 1. SDS-PAGE of Flavin Reductase from Bacillus sp. DSM411. DSM411 Lanes 1 and 7, marker proteins; lane 2, cell-free extract (10 g); We cultivated twenty-one thermophilic Bacillus lane 3, pooled fraction after Q-Sepharose (10 g); lane 4, pooled strains at 55 C, prepared cell-free extracts, and meas- fraction after Phenyl-Toyopearl (10 g); lane 5, pooled fraction after ured the enzyme activity to support DszC of R. eryth- FMN agarose (10 g); lane 6, purified enzyme after Superdex ropolis D-1. All strains had more or less coupling (10 g). activity. Since Bacillus sp. DSM411 showed not only relatively high specific reductase activity and high thermostability of the enzyme but also the best growth in terms of OD660 under the cultivation condition as Table 2. Substrate Specificity of Flavin Reductase described in ‘‘Materials and Methods’’ for the thermophilic strains tested, we selected this strain for the Compound Relative activity (%) subsequent experiments. Electron acceptora) Flavin reductase was purified 1,220-fold from Bacil- FMN 100 lus sp. DSM411. The purification steps are summarized FAD 61.4 Riboflavin 38.0 in Table 1. SDS-PAGE showed that the enzyme was Lumiflavin 31.5 purified to homogeneity and that the molecular mass of Nitrofurazone 1.7 the subunit was 16 kDa (Fig. 1). The native molecular Methyl 4-nitrobenzoate 2.0 mass of the enzyme was found to be 16 kDa by gel Nitrofurantoin 2.1 filtration, indicating that the flavin reductase is a 4-Nitroacetophenone 2.6 4-Nitrobenzoate 1.1 monomer enzyme. The purified enzyme solution did 4-Nitrotoluene 2.7 not show the yellow color typically derived from flavin 4-Nitrophenol 2.1 species. Menadione 3.2 1,4-Benzoquinone 3.2 Substrate specificity Methylene blue 6.7 Ferricyanide 2.1 Enzyme activity was measured using various electron Cytochrome c 2.4 acceptors in place of FMN (Table 2). When flavin DCPIP 4.9 compounds as electron acceptors were added at 20 M, Electron donorb) FAD, riboflavin, and lumiflavin were 62, 38, and 32% as NADH 100 effective as FMN respectively. Except for flavin com- NADPH 6.0 pounds, the enzyme acted on aromatic nitrocompounds a) The reaction mixture contained 20 mM potassium phosphate buffer (pH 7.0), such as nitrofurazone and 4-nitrotoluene and artificial 0.5 mM NADH, 20 M of each electron acceptor, and 0.54 g of the purified flavin reductase from the wild strain in a total volume of 0.5 ml. electron acceptors such as methylene blue and ferricya- b) The reaction mixture contained 0.5 mM NADH or NADPH and 20 M FMN nide, but these compounds were less than 10% as in a total volume of 0.5 ml. The other conditions were the same as above.a) 1716 T. OHSHIRO et al. Cloning of flavin reductase gene and its flanking region The N-terminal sequence of the enzyme was determined to be MDDRTFRRAMGKFATGVTVVTTEFQ- GEAKG. The sequence of the internal peptide fragment obtained with CNBr treatment was determined to be DEPGDPLLFFEGKYRSIGQ. Based on the sequences, degenerate primers FRNshi-5 and FRCshi-7 were designed, and PCR was done with total DNA of Bacillus sp. DSM411 as a template. Since the 450 bp fragment was amplified, it was introduced into the PCR cloning vector pGEM-T to construct pTFRS2. Sequencing of the Fig. 2. Time Course of DBT Monooxygenase Reaction with Purified DszC and Bfl. cloned DNA confirmed that it contained regions encod- The reaction mixture contained 100 mM potassium phosphate ing N-terminal and internal sequences of flavin reduc- buffer (pH 7.0), 0.27 mM DBT, 6 mM NADH, 10 M FMN, tase. Then, the probe was prepared by PCR using 0.216 mg/ml DszC, and 0.025 units/ml Bfl in a total volume of 0 pTFRS2 as a template, and the primers FRNshi-8, 5 - 250 l. The reaction was done at 40 C, and the amounts of DBT ATGGACGATCGGACGTTCCG-30 and FRCshi-10, 50- sulfone formed were determined as described in ‘‘Materials and 0 Methods’’. GGATCGCCGGGTTCGTCTTT-3 were designed. Southern hybridization was performed with total DNA of Bacillus sp. DSM411. Since a single band was effective as FMN. As an electron donor, the activity for detected in the 5.5 kb region when the total DNA was NADH was 17 times as high as that of NADPH. The Km double-digested with EcoRI and HindIII, the corre- values for NADH and FMN were 98.7 and 0.83 M sponding fragments were ligated into pBluescript II SK- respectively (data not shown). Excess amounts of FMN to construct the gene library. As a result of colony (above 300 M) inhibited enzyme activity (data not hybridization, five positive clones were obtained from shown). 1,200 colonies tested. One of the positive clones, pFREH12, was digested with SalI, and four fragments Coupling activity and ferric reductase activity (4.1, 2.2, 1.4, and 0.8 kb) were produced. Because only Coupling activity was examined between the present the 2.2 kb fragment was hybridized with the probe flavin reductase and DszC using both purified enzymes. described above, it was concluded that the fragment Formation of DBT sulfone was confirmed as shown in contained the flavin reductase gene. Hence subcloning Fig. 2, indicating that the flavin reductase from Bacillus was done with Sal1, and the resulting plasmid, pFRS5, sp. DSM411 coupled with the DszC activity in vitro. was sequenced (Fig. 4). An open reading frame (ORF) Previously, we purified two kinds of flavin reductases, was found within the 2.2 kb SalI fragment. It encoded a DszD from R. erythropolis D-125) and Flv from Paeni- protein of 155 amino acids with a molecular mass of bacillus polymyxa A-1.28) In this study we investigated 17,371 Da. The deduced N-terminal and internal amino the ferric reductase activities of three flavin reductases acid sequences from the ORF coincide with those of prepared in our laboratory. The enzyme reactions were flavin reductase. Hence this ORF was designated bfl done at 35 C for DszD, 55 C for Flv, and 65 C for Bfl (accession no. AB091771) for the thermophilic Bacillus respectively. As a result, Bfl and DszD had activities of flavin reductase gene. 601 and 52.5 units/mg respectively. In contrast, Flv A homology search revealed that the deduced bfl gene showed no activity. In the control experiments without product showed 59, 42, 36, and 30% amino acid identity FMN or ferricyanide, the decrease in absorbance at with PheA2, phenol 2-hydroxylase component B from 420 nm was negligible. Bacillus thermoglucosidasius (accession no. AF140605- 3);36) nitrilotriacetate monooxygenase component B General properties from Chelatobacter heintzii (accession no. I40751);37) The optimum temperature and pH of the enzyme were StyB, styrene monooxygenase component 2 from Pseu- 70 C and 6.0 respectively (Fig. 3A, B). The stability of domonas sp. (accession no. AJ000330-5);38) and HpaC, the enzyme was examined at various temperatures and 4-hydroxyphenylacetate 3-monooxygenase small com- pHs. The enzyme retained more than 90% of its activity ponent from E. coli (accession no. Z29081-3)39) respec- after it was incubated at 70 C for 30 min (Fig. 3C). It tively. Next, the original cloned 5.5 kb fragment in was also stable at pH 2–12 after incubation at 30 C for pFREH12 was sequenced, and three collinear and one 30 min (Fig. 3D). None of the metal ions tested elevated reverse ORFs were identified in addition to bfl (Fig. 5). enzyme activity (data not shown). Activity was inhibited A homology search clearly showed that ORF1 was by heavy metal compounds and p-chloromercuribenzoic similar to 5-carboxymethyl-2-hydroxymuconate semi- acid (data not shown), indicating that the SH group was aldehyde dehydrogenase from Brucella melitensis, with involved in the activity. 51% homology (accession no. AAL53376.1) and aldehyde dehydrogenase from Deinococcus radiodurans Flavin Reductase from a Thermophilic Bacterium 1717

Fig. 3. Effects of Temperature and pH on Flavin Reductase Activity and Stability. Enzyme reactions were carried out as described in ‘‘Materials and Methods’’ except that the reaction temperature (A) or the reaction buffer (B) varied. Enzyme activities were measured after the purified enzyme was preincubated at the various temperatures in 50 mM Tris–HCl buffer (pH 8.0) (C) and the various pHs at 30 C (D) for 30 min. In (B) and (D), the buffers used were citric acid-K2HPO4 ( ), potassium phosphate ( ), Tris–HCl ( ), and glycine-NaOH ( ). The total reaction volume was 0.5 ml, and 0.54 g of purified enzyme was added to each reaction mixture. The enzyme activity was determined by measuring the decrease in absorbance at 340 nm.

Fig. 4. Nucleotide and Deduced Amino Acid Sequences of Flavin Reductase from Bacillus sp. DSM411. The underlined sequences indicate N-terminal and internal amino acid sequences determined with the puriﬁed enzyme. The putative Shine- Dalgarno sequence is shown as a double underline. A broken line indicates the conserved motif, GDH of ﬂavin reductase involved in the interaction for NAD(P)H. with 50% homology (accession no. AAF12413.1). As and ORF4 were unknown based on a homology search for the three remaining ORFs, ORF2 was presumed to with the BLAST program. code a regulatory protein, and the functions of ORF3 1718 T. OHSHIRO et al.

Fig. 5. Physical Map of 5.5 kb-DNA Fragment Cloned from Bacillus sp. DSM411. Boxes show the localization of each open reading frame (ORF). Arrows indicate the orientation of the coding region.

Overexpression of bfl in E. coli this enzyme belongs to class I. But the recombinant Flavin reductase of Bacillus sp. DSM411 was over- enzyme preparation was yellow during the early stages produced in the recombinant E. coli. A DNA fragment of enzyme purification, suggesting the possibility that it containing bfl amplified by PCR was inserted into pET- bound flavins weakly. This phenomenon has also been 21a and the resulting plasmid was introduced into reported in the case of VlmR (a class I enzyme) from E. coli BL21(DE3). The level of expression of bfl by the Streptomyces viridifaciens.4) recombinant strain (101 units/mg) was 440-fold higher Some flavin reductases have been shown to catalyze than that by the wild strain, Bacillus sp. DSM411 the reduction not only of flavins such as FMN and FAD, (0.23 units/mg). Judging from the specific activity in the but also of nitroaromatic compounds.14–16) All these cell-free extracts, the recombinant E. coli cells ex- enzymes are flavoproteins. We have already investigated pressed as much as 40% of the total soluble protein. The the properties of two flavin reductases: DszD, a flavin flavin reductase was purified to homogeneity (three- reductase from R. erythropolis D-1 (a class I enzyme), fold) in a 14% yield from the recombinant strain (data had little activity for nitroaromatic compounds, and not shown). The enzyme solution was yellow derived NADH was specific as the electron donor,25) whereas from flavin species after a Q-Sepharose Fast Flow Flv, the enzyme from P. polymyxa A-1 (a class II column chromatography step, but the color disappeared enzyme), showed significant activity for nitroaromatic completely during the purification steps. compounds, and both NADH and NADPH served as preferred substrates.28) Flavin reductase from Bacillus Discussion sp. DSM411, Bfl, exhibited slight activity for some nitroaromatic compounds, and NADH was specific In this study, flavin reductase (Bfl) from the thermo- although NADPH showed very low activity as an philic Bacillus sp. DSM411 was purified and charac- electron donor. On the basis of these data, it is thought terized. The enzyme was thermostable and thermophilic. that Bfl has an intermediary substrate specificity be- Among many flavin reductases that have so far been tween the two enzymes described above, DszD and Flv. purified and characterized, the present flavin reductase, Furthermore, the enzyme was considered to bind a flavin Bfl, is the most thermophilic and thermostable. As for cofactor weakly. It might be of interest to investigate the TdsD from the thermophilic DBT-desulfurizing bacte- relationship between the binding of flavin and nitro- rium Paenibacillus sp. A11-2, it was reported that its reductase activity or reactivity for pyridine nucleotide optimum temperature was 55 C and that the remaining coenzymes. activity was 10% when TdsD was treated at 70 C for A homology search showed that Bfl had some 30 min.26) Recently, it was found that PheA2, which was similarities in amino acid sequence to the enzymes, the most similar to Bfl based on amino acid sequences, PheA2, StyB, and HpaC, that are involved in the had flavin reductase activity.40) The optimum temper- degradation of the aromatic compounds phenol, styrene, ature was 55 C and the remaining activity was 65% and 4-hydroxyphenylacetate respectively. Among these, when PheA2 was incubated at 65 C for 2 h. Bfl had PheA2 and HpaC have been confirmed to have flavin optimal activity at 70 C and retained 90% of the reductase activities.9,40) Recently, the flavin reductase activity even after the enzyme was treated at 70 C for gene was cloned from the thermophilic DBT-desulfur- 30 min (Fig. 3). Moreover, it was characteristic that the izing bacterium Bacillus subtilis WU-S2B by the unique enzyme did not lose its activity after it was treated at selection method.42) It is characteristic that indigo pH 2–12 for 30 min. production by coexpression of the DBT monooxygenase The two major classes of flavin reductases have been gene from B. subtilis WU-S2B in E. coli was used as an proposed to date:41) class I enzymes do not contain any indication for the selection. The deduced amino acid flavin prosthetic group, whereas class II enzymes are sequence of this gene had 61% identity with that of the typical flavoproteins. The purified flavin reductase from nitroreductase (YwrO) of B. amyloliquefaciens. Galań et Bacillus sp. DSM411 did not show the yellow color al. aligned several flavin reductases by amino acid typical of flavin-containing enzymes, suggesting that sequences and proposed a new flavin reductase sub- Flavin Reductase from a Thermophilic Bacterium 1719 group. DszD, StyB, and HpaC belonged to this group on References their classification.9) They suggested that flavin reductases belonging to this group contain a highly conserved 1) Tu, S.-C., and Mager, H. I. X., Biochemistry of bacterial GDH motif in the C terminal region, which might play a bioluminescence. Photochem. Photobiol., 62, 615–624 role in NAD(P)H interaction.9) Bfl also has this motif (1995). 2) Kendrew, G. S., Harding, S. E., Hopwood, D. A., and sequence (Fig. 4), suggesting that it belongs to this Marsh, E. N. G., Identification of a flavin:NADH family. oxidoreductase involved in the biosynthesis of actino- The flavin reductases from R. erythropolis D-1, rhodin. J. Biol. Chem., 270, 17339–17343 (1995). Paenibacillus sp. A11-2, and P. polymyxa A-1 are 3) Thibaut, D., Ratet, N., Bisch, D., Faucher, D., known to couple with monooxygenases involved in Debussche, L., and Blanche, F., Purification of the microbial desulfurization. Bfl showed no similarity in two-enzyme system catalyzing the oxidation of the D- amino acid sequence to such flavin reductases. Never- proline residue of pristinamycin IIB during the last step theless, it was confirmed that the purified Bfl supported of pristinamycin IIA biosynthesis. J. Bacteriol., 177, DszC activity in vitro, as shown in Fig. 2. The reduced 5199–5205 (1995). flavins produced by flavin reductase are known to 4) Parry, J. R., and Li, W., An NADPH:FAD oxidoreduc- support many monooxygenases. Although it is necessary tase from valanimycin producer, Streptomyces viridifaciens. J. Biol. Chem., 272, 23303–23311 (1997). to examine the coupling activities with DszA and other 5) Witschel, M., Nagel, S., and Egli, T., Identification and monooxygenases, we expect that Bfl may be applicable characterization of the two-enzyme system catalyzing not only in microbial desulfurization but also in oxidation of EDTA in the EDTA-degrading bacterial monooxygenase systems in aromatic compounds degra- strain DSM 9103. J. Bacteriol., 179, 6937–6943 (1997). dation at high temperature. 6) Xu, Y., Mortimer, M. W., Fisher, T. S., Kahn, M. L., The gene most similar to bfl was pheA2, which is one Brockman, F. J., and Xun, L., Cloning, sequencing and component in the phe operon for phenol degradation of analysis of a gene cluster from Chelatobacter heintzii the thermophilic bacterium B. thermoglucosidasius ATCC 29600 encoding nitrilotriacetate monooxygenase A7.36) The gene coding the large component of the and NADH:flavin mononucleotide oxidoreductase. J. phenol hydroxylase pheA1, which was located 20 bp Bacteriol., 179, 1112–1116 (1997). upstream of pheA2, and other genes involved in the 7) Eichhorn, E., van der Ploeg, J. R., and Leisinger, T., Characterization of a two-component alkanesulfonate phenol degradation were confirmed within the phe monooxygenase from Escherichia coli. J. Biol. Chem., operon of this strain. In contrast, no genes for degrada- 274, 26639–26646 (1999). tion of aromatic compounds were found around bfl 8) Kertesz, M., Schmidt-Larbig, K., and Wuest, T., A novel except for ORF1, which was similar to aldehyde reduced flavin mononucleotide-dependent methanesul- dehydrogenase and located upstream of it. Although it fonate sulfonatase encoded by the sulfur-regulated msu is necessary to investigate further to make clear the operon of Pseudomonas aeruginosa. J. Bacteriol., 181, functions of ORF2, 3, and 4, it is thought that Bfl is not 1464–1473 (1999). an enzyme that couples with a specific monooxygenase. 9) Galań, B., Dıáz, E., Preto, M. A., and Garcıá, J. L., In 1999, it was shown that ferric reductase (FeR) from Functional analysis of the small component of the 4- a hyperthermophilic archeon, Archaeoglobus fulgidus, hydroxyphenylacetate 3-monooygenase of Escherichia shared amino acid similarity with a family of flavin coli W: a prototype of a new flavin:NAD(P)H reductase subfamily. J. Bacteriol., 182, 627–636 (2000). reductase,33) and the three-dimensional structures of FeR 40,43) 10) Xun, L., and Sandvik, E. R., Characterization of 4- and PheA2 were elucidated and compared Never- hydroxyphenylacetate 3-hydroxylase (HpaB) of Esche- theless, it was reported that FeR had flavin reductase richia coli as a reduced flavin adenine dinucleotide- activity and that PheA2 had no ferric reductase utilizing monooxygenase. Appl. Environ. Microbiol., 66, activity.40) In this study we confirmed that Bfl had ferric 481–486 (2000). reductase activity. It should be interesting to elucidate 11) Zenno, S., and Saigo, K., Identification of the genes whether this activity might be related to the physiolog- encoding NAD(P)H-flavin oxidoreductases that are ical role of Bfl. Further studies are required to clarify the similar in sequence to Escherichia coli Fre in four true function of flavin reductases whose genes are not in species of luminous bacteria: Photorhabdus lumines- the gene cluster for the metabolism of specific com- cens, Vibrio fischeri, Vibrio harveyi, and Vibrio orien- pounds. talis. J. Bacteriol., 176, 3544–3551 (1994). 12) Zenno, S., Saigo, K., Kanoh, H., and Inouye, S., Identification of the gene encoding the major Acknowledgments NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744. J. Bacteriol., 176, Part of this work was conducted with the support of 3536–3543 (1994). the Japan Cooperation Center, Petroleum (JCCP). It was 13) Koike, H., Sasaki, H., Kobori, T., Zenno, S., Murphy, M. also supported by Grant-in-Aid for Scientific Research E. P., Adman, E. T., and Tanokura, M., 1.8 A crystal 15580063 (to Y. I.) from the Ministry of Education, structure of the major NAD(P)H:FMN oxidoreductase of Culture, Sports, Science, and Technology of Japan. a bioluminescent bacterium, Vibrio fischeri: Overall structure, cofactor and substrate-analog binding, and 1720 T. OHSHIRO et al. comparison with related flavoproteins. J. Mol. Biol., 280, and Maruhashi, K., Purification and characterization of 259–273 (1998). dibenzothiophene sulfone monooxygenase and FMN- 14) Zenno, S., Koike, H., Kumar, A. N., Jayaraman, R., dependent NADH oxidoreductase from the thermophilic Tanokura, M., and Saigo, K., Biochemical character- bacterium Paenibacillus sp. strain A11-2. J. Biosci. ization of NfsA, the Escherichia coli major nitroreduc- Bioeng., 90, 607–613 (2000). tase exhibiting a high amino acid sequence homology to 27) Ishii, Y., Konishi, J., Suzuki, M., and Maruhashi, K., Frp, a Vibrio harveyi flavin oxidoreductase. J. Bacteriol., Cloning and expression of the gene encoding the 178, 4508–4514 (1996). thermophilic NAD(P)H-FMN oxidoreductase coupling 15) Zenno, S., Koike, H., Tanokura, M., and Saigo, K., Gene with the desulfurization enzymes from Paenibacillus sp. cloning, purification, and characterization of NfsB, a A11-2. J. Biosci. Bioeng., 90, 591–599 (2000). minor oxygen-insensitive nitroreductase from Esche- 28) Ohshiro, T., Aoi, Y., Torii, K., and Izumi, Y., Flavin richia coli, similar in biochemical properties to FRaseI, reductase coupling with two monooxygenases involved the major flavin reductase in Vibrio fischeri. J. Biochem., in dibenzothiophene desulfurization: purification and 120, 736–744 (1996). characterization from a non-desulfurizing bacterium, 16) Zenno, S., Kobori, T., Tanokura, M., and Saigo, K., Paenibacillus polymyxa A-1. Appl. Microbiol. Biotech- Purification and characterization of NfrA1, a Bacillus nol., 59, 649–657 (2002). subtilis nitro/flavin reductase capable of interacting with 29) Ishii, Y., Ohshiro, T., Aoi, Y., Suzuki, M., and Izumi, Y., bacterial luciferase. Biosci. Biotechnol. Biochem., 62, Identification of the gene encoding a NAD(P)H-flavin 1978–1987 (1998). oxidoreductase coupling with dibenzothiophene (DBT)- 17) Ingelman, M., Ramasswamy, S., Nivie´re, V., Fontecave, desulfurizing enzyme from the DBT-nondesulfurizing M., and Eklund, H., Crystal structure of NAD(P)H:flavin bacterium Paenibacillus polymyxa A-1. J. Biosci. Bio- oxidoreductase from Escherichia coli. Biochemistry, 38, eng., 90, 220–222 (2000). 7040–7049 (1999). 30) Konishi, J., Ishii, Y., Onaka, T., Okumura, K., and 18) Kobori, T., Sasaki, H., Lee, W. C., Zenno, S., Saigo, K., Suzuki, M., Thermophilic carbon-sulfur-bond-targeted Murphy, M. E. P., and Tanokura, M., Structure and site- biodesulfurization. Appl. Environ. Microbiol., 63, 3164– directed mutagenesis of a flavoprotein from Escherichia 3169 (1997). coli that reduces nitrocompounds. J. Biol. Chem., 276, 31) Furuya, T., Kirimura, K., Kino, K., and Usami, S., 2816–2823 (2001). Thermophilic biodesulfurization of dibenzothiophene 19) Ohshiro, T., and Izumi, Y., Microbial desulfurization of and its derivatives by Mycobacterium phlei WU-F1. organic sulfur compounds in petroleum. Biosci. Bio- FEMS Microbiol. Lett., 204, 129–133 (2001). technol. Biochem., 63, 1–9 (1999). 32) Kirimura, K., Furuya, T., Nishii, Y., Ishii, Y., Kino, K., 20) Ohshiro, T., and Izumi, Y., Desulfurization of fossil and Usami, S., Biodesulfurization of dibenzothiophene fuels. In ‘‘Encyclopedia of Environmental Microbiol- and its derivatives through the selective cleavage of ogy’’, ed. Bitton, G., John Wiley & Sons, Inc., New carbon-sulfur bonds by a moderately thermophilic York, pp. 1041–1051 (2002). bacterium Bacillus subtilis WU-S2B. J. Biosci. Bioeng., 21) Ohshiro, T., Suzuki, K., and Izumi, Y., Dibenzothio- 91, 262–266 (2001). phene (DBT) degrading enzyme responsible for the first 33) Vadas, A., Monbouquette, H. G., Johnson, E., and step of DBT desulfurization by Rhodococcus erythro- Schro¨der, I., Identification and characterization of a polis D-1: purification and characterization. J. Ferment. novel ferric reductase from the hyperthermophilic Bioeng., 83, 233–237 (1997). archaeon Archaeoglobus fulgidus. J. Biol. Chem., 274, 22) Ohshiro, T., Kojima, T., Torii, K., Kawasoe, H., and 36715–36721 (1999). Izumi, Y., Purification and characterization of dibenzo- 34) Sambrook, J., and Russell, D. W., Molecular cloning: a thiophene (DBT) sulfone monooxygenase, an enzyme laboratory manual, 3rd ed., Cold Spring Harbor Labo- involved in DBT desulfurization, from Rhodococcus ratory Press, Cold Spring Harbor, N.Y. (2001). erythropolis D-1. J. Biosci. Bioeng., 88, 610–616 (1999). 35) Bradford, M. M., A rapid and sensitive method for 23) Nakayama, N., Matsubara, T., Ohshiro, T., Moroto, Y., quantitation of microgram quantities of protein utilizing Kawata, Y., Koizumi, K., Hirakawa, Y., Suzuki, M., the principle of protein-dye binding. Anal. Biochem., 72, Maruhashi, K., Izumi, Y., and Kurane, R., A novel 248–254 (1976). enzyme 20-hydroxybiphenyl-2-sulfinate desulfinase 36) Duffiner, M. F., Kirchner, U., Bauer, M. P., and Mu¨ller, (DszB), from a dibenzothiophene-desulfurizing bacteri- R., Phenol/cresol degradation by the thermophilic um Rhodococcus erythropolis KA2-5-1: gene overex- Bacillus thermoglucosidasius A7: cloning and sequence pression and enzyme characterization. Biochim. Biophys. analysis of five genes involved in the pathway. Gene, Acta, 1598, 122–130 (2002). 256, 215–221 (2000). 24) Xi, L., Squires, C. H., Monticello, D. J., and Childs, 37) Uetz, T., Schneider, R., Snozzi, M., and Egli, T., J. D., A flavin reductase stimulates DszA and DszC Purification and characterization of a two-component proteins of Rhodococcus erythropolis IGTS8 in vitro. monooxygenase that hydroxylates nitrilotriacetate from Biochem. Biophys. Res. Commun., 230, 73–75 (1997). ‘Chelatobacter’ strain ATCC 29600. J. Bacteriol., 174, 25) Matsubara, T., Ohshiro, T., Nishina, Y., and Izumi, Y., 1179–1188 (1992). Purification, characterization and overexpression of 38) Velasco, A., Alonso, S., Garcıá, J. L., Perera, J., and flavin reductase involved in dibenzothiophene desulfur- Dıáz, E., Genetic and functional analysis of the styrene ization by Rhodococcus erythropolis D-1. Appl. Environ. catabolic cluster of Pseudomonas sp. strain Y2. J. Microbiol., 67, 1179–1184 (2001). Bacteriol., 180, 1063–1071 (1998). 26) Konishi, J., Ishii, Y., Onaka, T., Ohta, Y., Suzuki, M., 39) Prieto, A., Marıá, E., Dıáz, E., and Garcıá, J. L., Flavin Reductase from a Thermophilic Bacterium 1721 Molecular characterization of the 4-hydroxyphenylace- richia coli. J. Biol. Chem., 274, 18252–18260 (1999). tate catabolic pathway of Escherichia coli W: engineer- 42) Furuya, T., Takahashi, S., Ishii, Y., Kino, K., and ing a mobile aromatic degradative cluster. J. Bacteriol., Kirimura, K., Cloning of a gene encoding flavin 178, 111–120 (1996). reductase coupling with dibenzothiophene monooxyge- 40) Kirchner, U., Westphal, A. H., Mu¨ller, R., and van nase through coexpression screening using indigo pro- Berkel, W. J. H., Phenol hydroxylase from Bacillus duction as selective indication. Biochem. Biophys. Res. thermoglucosidasius A7, a two-protein component mon- Commun., 313, 570–575 (2004). ooxygenase with a dual role for FAD. J. Biol. Chem., 43) Chiu, H.-J., Johnson, E., Schro¨der, I., and Rees, D. C., 278, 47545–47553 (2003). Crystal structures of a novel ferric reductase from the 41) Nivie´re, V., Fieschi, F., Dećout, J.-L., and Fontecave, hyperthermophilic archaeon Archaeoglobus fulgidus and M., The NAD(P)H:Flavin oxidoreductase from Esche- its complex with NADPþ. Structure, 9, 311–319 (2001).