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 monooxyge- nases 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 Â 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 Â 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 Â 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 Â g and stored 0.5 mM NADH, 20 M FMN, and the enzyme in a total at À20 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 Â 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.