Báo cáo Y học: Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes pot

Cloning, expression and characterization of a gene encoding nitroalkane-oxidizing enzyme from Streptomyces ansochromogenes Jihui Zhang, Wenbo Ma* and Huarong Tan Institute of Microbiology, Chinese Academy of Sciences, Beijing, China A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library of Streptomyces ansochromo- genes 7100. The deduced protein (NaoA) of this gene con- tains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organ- isms. The naoA gene was subcloned into an expression vector pET23b and overexpressed in Escherichia coli BL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can con- vert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds. The optimal pH and temperature for NaoA was found to be pH 7–8 and 48–56 °C, respectively. The K m of NaoA for nitroethane is  26.8 m M . NADH and nitro blue tetrazolium are strong inhibitors of NaoA, and thiol compounds and superoxide dismutase partially inhibit the enzyme activity. Therefore, superoxide may be an essential intermediate in the oxidation of nitroalkane by NaoA. Keywords: enzymatic properties; expression; gene cloning; nitroalkane-oxidizing enzyme; Streptomyces. Nitroalkane compounds are widely used in chemical indus- try as intermediates, solvents and fuel for rockets [1] and are released in large quantities into the environment. Mean- while, certain micro-organisms and many leguminous plants produce nitroalkane compounds [2]. These materials are hazardous and can result in environmental contamination. So the conversion of nitro groups by biocatalysts is useful in industry as well as in environmental conservation. Enzymes that can convert nitroalkanes into less harmful species have been purified and characterized from micro-organisms. They include 2-nitropropane dioxygenase from Williopsis satur- mus var. mrakii [3–5] and Neurospora crassa [6,7] and nitroalkane oxidase from Fusarium oxysporum [8] and Aspergillus flavus [9]. The conversion of nitro compounds into less harmful materials by nitroalkane-oxidizing enzymes in organisms may have the physiological significance of inactivating the natural defenses of plants [10]. The reaction mechanisms of several nitroalkane-oxidizing enzymes have been analyzed [6,11,12]. Thus, 2-nitropropane dioxygenase from W.saturmusvar. mrakii catalyzes the incorporation of two atoms of oxygen molecule into two molecules of the same acceptor, and the enzyme is an intermolecular dioxyg- enase [4], and nitroalkane oxidase from F. oxysporum has a hydrophobic microenvironment of the flavin cofactor [11,13]. The genes encoding 2-nitropropane dioxygenase from W.saturmusvar. mrakii and nitroalkane oxidase were cloned and expressed in Escherichia coli [14,15]. Dhawale et al. [16] reported that crude cell-free extracts of Streptomyces could catalyze the oxidation of nitroalkanes to form carbonyl compounds and nitrite, but the genes related to these enzymes in Streptomyces have not been reported so far. Our previous experiments revealed that DNA upstream of P TH270 , a differentiation-related promoter in Streptomyces [17,18], contained an incomplete ORF the deduced product of which had high similarity to 2-nitropropane dioxygenase from W.saturmusvar. mrakii. This led to the experiment to identify whether the protein encoded by the complete DNA fragment can catalyze the oxidation of nitroalkanes. In this paper, we describe the cloning and characterization of a novel gene (naoA) that encodes nitroalkane-oxidizing enzyme in S. ansochromogenes. MATERIALS AND METHODS Strains, plasmids and growth conditions S. ansochromogenes 7100 [19], E. coli JMl09, BL21(DE3) [20], pBluescript Ml3 – , pET23b (Novagen) and M13 KO7 [21] as the helper phage were collected in this laboratory. pIJ4477 was constructed during the work described in [18]; pTH1104 (Ml3 – containing naoA) and pNA101 (pET23b containing naoA) were constructed in this work. S. anso- chromogenes mycelium was grown in yeast extract/malt extract liquid medium on a rotary shaker at 28 °C[22]. JMl09 and BL21(DE3) strains were grown at 37 °C, in Luria–Bertani medium supplemented with 100 lgÆmL )1 ampicillin when necessary [23]. DNA manipulations Plasmid and chromosomal DNA was isolated from Streptomyces or E. coli by established techniques [22,23]. Correspondence to H. Tan, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China. Fax: + 86 10 62654083, Tel.: + 86 10 62654083, E-mail: tanhr@sun.im.ac.cn Abbreviations: naoA, nitroalkane-oxidizing enzyme gene A; MBTH, 3-methyl benzothiazolone hydrazone hydrochloride. Note: the nucleotide sequence of naoA gene has been deposited in GenBank under the accession number AF284037. *Present address:DepartmentofBiology,UniversityofWaterloo, Waterloo, ON N2L 3G1. Canada. (Received 7 August 2002, revised 25 October 2002, accepted 5 November 2002) Eur. J. Biochem. 269, 6302–6307 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03350.x Transformation of E. coli strains, Southern blotting, and colony hybridization were carried out as described by Sambrook et al. [23]. Restriction enzymes and T4 DNA ligase were purchased from Boehringer-Mannheim and Sino-American Biotechnology Company (Luoyang, Chi- na) 1 . DIG labeling and detection kits (Boehringer-Mann- heim) were used for preparation of DNA probes according to the protocols of the manufacturer. DNA sequencing and analysis Plasmid pTH1104 containing the target fragment was digested with exonuclease III by the reported method [24] to generate a set of nested deletions from each end of the inserts. Appropriately deleted derivatives were sequenced by the dideoxy chain termination method using the Taq Track sequencing kit (Promega, Madison, WI, USA) and [a- 32 P]dCTP as the labeled nucleotide. ORF analysis was based on the specific codon usage of Streptomyces [25]. Deduced amino-acid sequence was compared with the database in the National Center for Biotechnology Information (NCBI) using the Basic BLAST search [26]. Primers and PCR conditions To achieve overexpression of naoA, two primers were designed from the complete DNA sequence of naoA (P1, 5¢-GA CATATGTCCTCCGCGCTGA-3¢;P2,5¢-GGAA GCTTTCACCCCTTACGGGA-3¢,withNdeIandHindIII restriction sites underlined). Pfu DNA polymerase (Sangon Co., Shanghai, China) was used to amplify naoA,with pTH1104 as template. The following PCR program was performed: an initial denaturalization at 95 °Cfor5min followed by 30 cycles of amplification (95 °Cfor1min, 55 °C for 1 min, and 72 °C for 1 min) and an additional extensionstepat72°C for 10 min. The PCR product (about 1.1 kb) was purified by agarose gel electrophoresis and then subcloned into the NdeIandHindIII sites of pET23b for overexpression. Electrophoresis of proteins SDS/PAGE was carried out as described previously [23]. IEF was performed using a Computer Controlled Electro- phoresis Power Supply and a Mini IEF Cell (Bio-Rad). Protein standards of different pI were as follows: amylo- glucosidase, pI 3.6; b-lactoglobulin A, pI 5.1; myoglobin, pI 6.8/7.2; trypsinogen, pI 9.3. The pI of NaoA was determined from a standard curve of pI and migration distance (cm) of protein standards. Enzyme assay and analytical methods E. coli BL21(DE3) containing plasmid pNA101 was grown at 37 °C for 12–14 h or overnight in Luria–Bertani medium supplemented with ampicillin (100 lgÆmL )1 ), and then 40 mL Luria–Bertani medium was inoculated with 40 lL of the above fresh overnight culture and incubated at 37 °C with shaking until cells were grown to D 600 ¼ 0.4, usually about 2.5–3 h. The culture was induced (1 m M isopropyl thio-b- D -galactoside) and grown for a further 3 h, and then the cells were harvested by centrifugation at 10 000 g for 3 min and suspended in 100 m M sodium phosphate buffer (pH 8.0). A 10-mL volume of cell suspension was discontinuously sonicated (100 W; JY96- II sonicator) for 5 min on ice to generate cell extracts after centrifugation at 10 000 g for 3 min. Protein concentra- tions were determined by the Biuret reaction using BSA as standard [27]. Activity of NaoA was detected with nitroethane, 1-nitropropane, 2-nitropropane or nitrometh- ane as substrate. The standard reaction mixture consisted of 4 m M nitroalkane, cell extract and 0.1 M sodium phosphate (pH 8.0 for 2-nitropropane and pH 7.0 for 1- nitropropane or nitroethane) and was finally maintained in a 0.6-mL volume. Nitrite released from the reaction was determined by the method of Little [28]. One unit of NaoA is defined as the amount of enzyme required to catalyze the formation of 1 lmol nitriteÆmin )1 . Carbonyl compounds (aldehyde and ketone) can react with 3-methyl benzothi- azolone hydrazone hydrochloride (MBTH; purchased from the Fluka Chemical Company) to form azines, which display a characteristic absorbance peak at 304–310 nm. Therefore, formation of carbonyl compounds from nitro- alkanes can be demonstrated with MBTH using an improved method [29,30]. The reaction of the aldehyde group with ferric chloride was used to detect them during this study, and acetone was determined with GC-MS on the Shimadzu GCMS-QP5050A. Acetaldehyde and acet- one were used as standards. Purification of NaoA Cells of BL21(DE3) carrying pNA101 were harvested by centrifugation (10 000 g, 3 min). After suspension in 100 m M sodium phosphate buffer (pH 8.0), they were sonicated and centrifuged at 10 000 g for 30 min to remove cell debris. NaoA was purified from the supernatant according to the following steps. Step 1: Solid ammonium sulfate was added to the crude extracts to a final concentration of 10% saturation, and the precipitate was removed by centrifugation. Then, solid ammonium sulfate was added to the supernatant to give 80% saturation. The resulting precipitate containing the enzyme activity was collected by centrifugation at 10 000 g for30minandthendissolvedin10m M sodium phosphate buffer (pH 8.0). Step 2: The crude protein solution was loaded onto a Sephadex G75 column pre-equilibrated with 10 m M sodium phosphate buffer (pH 8.0) and eluted with the same buffer. The active fractions were collected. Step 3: NaCl was added to the pooled active fractions to a final concentration of 0.2 M .Then,theproteinsolutionwas run on a DEAE-Sepharose Fast Flow column pre-equili- brated with 10 m M sodium phosphate buffer (pH 8.0). The column was washed with 0.2 M NaCl until no protein was eluted, and then bound NaoA was eluted with 0.35 M NaCl inthesamebuffer. Step 4: After being desalted with an ultrafiltration tube (Pall Corporation), the active fractions were further run on a DEAE-Sepharose Fast Flow column pre-equilibrated with 10 m M sodium phosphate buffer (pH 8.0). The column was first washed with the same buffer containing 0.2 M NaCl, and then the proteins were eluted with a 0.2–0.4 M NaCl gradient in the above buffer (flow rate, 0.4 mLÆmin )1 ). Ó FEBS 2002 A nitroalkane-oxidizing enzyme of Streptomyces (Eur. J. Biochem. 269) 6303 Experiments on NaoA properties The enzymatic reaction was assayed in sodium phosphate buffer (0.1 M ) at different pH values and temperatures to define optimal conditions. Various compounds were also investigated for their inhibitory effects on enzyme activity. Superoxide dismutase was purchased from Sigma Chemical Company; its unit of activity was as defined by the manufacturer. Different concentrations of nitroethane were used to test the relationship between initial velocity and substrate concentration. Velocity was determined by detecting the formation of nitrite using the method of Ida et al.[31].K m and V max of purified NaoA were determined from a double-reciprocal plot according to the Lineweaver- Burk equation [32]. RESULTS Cloning of 1.5-kb DNA fragment A 320-bp DNA fragment located upstream of P TH270 (a differentiation-related promoter of Streptomyces coeli- color) was obtained by digesting pIJ4477 with SmaIand HindIII. This DNA fragment was labeled with the digoxigenin-11-dUTP kit (Roche, Mannheim, Germany) and used as a probe for Southern-blot hybridization with the digested genomic DNA of S. ansochromogenes. Approximately 7.0-kb DNA fragments with a positive signal were separated from the genomic DNA digested with NotI by agarose gel electrophrosis, and then a partial DNA library was constructed in E. coli JM109 using pBluescript M13 – as vector. The library was screened by colony hybridization using the above probe. Several positive colonies were identified and confirmed by Southern-blot hybridization (data not shown). The recombinant plasmid was further digested with SstII, and a 1.5-kb DNA fragment still displayed a strong positive signal after hybridization. DNA sequencing analysis DNA sequencing analysis showed that the 1.5-kb DNA fragment contains one complete ORF with 1092 nucleo- tides. The overall G + C content is 74%, which is typical for genes of Streptomyces. A potential ribosome-binding site (GGAAGGA) was located at the 18–24 base positions from the start codon (ATG). The deduced protein had a molecular mass of  37 kDa and showed identity (Blast output) with the following proteins in database searches (Fig. 1): 78% with the putative oxidoreductase from Streptomyces coelicolor, 27% with 2-nitropropane dioxyg- enase of W.saturmusvar. mrakii, 26% with 2-nitropropane dioxygenase of N. crassa, and 36% with the putative 2-nitropropane dioxygenase encoded by yrpB of Bacillus subtilis. Therefore, the gene product may be involved in the degradation of nitroalkanes and this gene was designated naoA (nitroalkane-oxidizing enzyme gene). Expression of naoA in E. coli To study the function of the naoA gene, it is necessary to obtain an adequate amount of NaoA protein. Therefore, the naoA gene was subcloned into pET23b to generate plasmid pNA101, and then it was introduced into BL21(DE3) for high-level expression under the control of the T7 promoter. After induction with isopropyl thio- b- D -galactoside, a 37-kDa protein band from the extracts of BL21(DE3)/pNA101 appeared on SDS/PAGE, whereas no protein bands from the extracts of BL21(DE3)/pET23b as control were found at the same position on SDS/PAGE (Fig. 2). The result indicated that the naoA gene was efficiently expressed in E. coli. Purification and characterization of NaoA Protein extracts of BL21(DE3)/pNA101 were separated by gel filtration, anion-exchange column chromatography, and ultrafiltration. The purified NaoA was further detected by SDS/PAGE (Fig. 2). The data for NaoA purification are summarized in Table 1. The specific activity of the purified NaoA was about 21 times higher than that of crude extract, and the yield was 34%. The relative activities of purified NaoA with nitroethane, 1-nitropropane and 2-nitropropane were, respectively, 100%, 90.7%, 5.89% when the substrate concentration was 4 m M . Reaction mixtures containing the purified NaoA and substrate (1-nitropropane, 2-nitropropane or nitroethane) were incubated for 5 min at 37 °C. After the reaction solution was mixed with o-aminophenylsulfuric acid and a-naphthanamine solutions, a red color appeared. The cell extracts of BL21(DE3)/pET23b as control did not show a coloring reaction, indicating that 1-nitropropane, Fig. 1. Comparison of NaoA with other nitroalkane-oxidizing enzymes. SA, NaoA from S. ansochromogenes; SC, putative 2-nitropropane dioxygenase from S. coelicolor; WS, 2-nitropropane dioxygenase from W.saturmus var. mrakii; NC, 2-nitropropane dioxygenase from N. crassa; BS, 2-nitropropane dioxygenase-related protein encoded by yrpB gene from Bacillus subtilis. Amino-acid residues with high iden- tity are shaded. The program OMIGA 2.0 was used to compare amino- acid sequences. 6304 J. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002 2-nitropropane and nitroethane can be oxidized and deni- trified to form nitrite by NaoA. Furthermore, it was clear that nitromethane was not a substrate of NaoA because no red color, which would have indicated the production of nitrite, was seen in assays containing nitromethane (Fig. 3). In addition to nitrite formation, carbonyl compounds released in the oxidation of nitroalkanes by NaoA were determined with MBTH. After reaction with MBTH, the UV spectra of the reaction solutions with nitroethane, 1-nitropropane or 2-nitropropane had a typical maximum absorption at 304–310 nm, which was identical with that of the expected carbonyl products reacted with MBTH. A deep green color was obtained after reaction with FeCl 3 using nitroethane or 1-nitropropane as substrate (maximum peak 640–670 nm). This result showed that the correspond- ing aldehydes were formed during the oxidation of nitro- ethane or 1-nitropropane in the presence of NaoA. When 2-nitropropane was used as substrate, the carbonyl com- pound formed in the reaction solution was further con- firmed to be acetone by its mass spectrum, which displayed fragments of m/z 58 (M + )andm/z 43 consistent with those of acetone standard. Properties of NaoA The pI of NaoA is about 5.2 according to the standard plot between the protein’s pI and its migration distance (cm) in IEF. The optimal pH and temperature of purified NaoA were 7.0–8.0 (data not shown) and 48–56 °C, respectively, in 0.1 M sodium phosphate buffer. NaoA activity increased over the temperature range 20–50 °C but declined rapidly above 60 °C. The effects of various compounds on NaoA activity were also examined (Table 2). Mn 2+ increased the enzyme activity slightly, and Cu 2+ inhibited it. Mg 2+ and Ca 2+ did not affect NaoA activity. Thiol groups may be involved in the active site of NaoA because thiol compounds (2-mercaptoethanol, GSH) partially inhibited activity. Unlike the nitroalkane oxidase from F. oxysporum [8], NADH strongly decreased the NaoA activity. NaoA is almost completely inactive in the presence of the super- oxide-scavenging agent nitro blue tetrazolium at a concen- tration of 5 m M . When the amount of superoxide dismutase reached 200 U, the relative activity of NaoA remained 6.1% and 51%, respectively, with 2-nitropropane and nitroethane as substrate. These results suggest that superoxide anion radicals are essential intermediates in the oxidation of nitroalkane by NaoA. The K m of purified NaoA for nitroethane was found to be  26.8 m M ,andV max for the formation of nitrite 0.175 lmolÆmin )1 Ælg )1 according to the Lineweaver-Burk equation. DISCUSSION We have cloned and determined the complete sequence of a gene encoding nitroalkane-oxidizing enzyme from Strepto- myces; partial purification of the related enzyme from Streptomyces has been reported [16]. The deduced amino- acid sequence of NaoA from S. ansochromogenes has high identity with that of the putative oxidoreductase from S. coelicolor [33,34]. The two proteins consist of 363 and 364 residues, respectively. Moreover, the consensus sequence Fig. 2. SDS/PAGE of NaoA expressed in E. coli and its purification. Lane 1, total protein from BL21(DE3)/pET23b; lane 2, total protein from strain BL21(DE3)/pNA101; lane 3, recombinant NaoA after 80% ammonium sulfate fraction; lane 4, recombinant NaoA after Sephadex G75 chromatography; lane 5, purified recombinant NaoA after DEAE-Sepharose Fast Flow chromatography; lane 6, standard molecular mass markers (phosphorylase b,97kDa;BSA,66kDa; ovalbumin, 45 kDa). Fig. 3. Assay of NaoA activity. (A) Protein extracts from BL21(DE3)/ pET23b. (B) Protein extracts from BL21(DE3)/pNA101; lane 1, sub- strate 2-nitropropane; lane 2, substrate nitroethane; lane 3, substrate 1-nitropropane; lane 4, substrate nitromethane. 2 m M substrate and 100 lL protein extracts were used in the reaction. Table 1. Purification of NaoA from E. coli. The activity is measured according to the formation of nitrite using 1-nitropropane as substrate. Purification step Total protein (mg) Total activity (U) Specific activity [UÆ(mg protein) )1 ] Purification (fold) Yield (%) Cell extract 307 1860 6 1.00 100 Sephadex G75 172 1756 10 1.7 94 DEAE-Sepharose Fast Flow 5 633 127 21 34 Ó FEBS 2002 A nitroalkane-oxidizing enzyme of Streptomyces (Eur. J. Biochem. 269) 6305 GXGXXA, which exists in many nucleotide-binding domains of dehydrogenases [35], was found at positions 36–41 in the deduced NaoA protein (GSGFLA) as well as in the putative oxidoreductase of S. coelicolor (GLGFLA). NaoA also displayed features that resemble those of 2-nitropropane dioxygenase from W. saturmus var. mrakii and those of nitroalkane oxidase from F. oxysporum. Both carbonyl compounds and nitrite, the common products of nitroalkane oxidation catalyzed by 2-nitropropane dioxyg- enase and nitroalkane oxidase, were detected in the oxidation of 1-nitropropane, 2-nitropropane and nitro- ethane catalyzed by NaoA, indicating that NaoA is a type of nitroalkane-oxidizing enzyme. Furthermore, the deduced amino-acid sequence of NaoA has higher identity with those of 2-nitropropane dioxygenase characterized in W. saturmus var. mrakii [14], and the inhibitory effects of various compounds on NaoA activity are also similar to 2-nitropropane dioxygenase. Therefore, NaoA is possibly a nitroalkane dioxygenase-like enzyme. The enzymatic properties of NaoA are a little different from those of other nitroalkane dioxygenases. The K m of NaoA for nitroethane (26.8 m M ) is similar to that of 2-nitropropane dioxygenase [4,6], but is quite different from that of nitroalkane oxidase from F. oxysporum (1 m M )[8].From the substrate specificity, 2-nitropropane is the preferred substrate for 2-nitropropane dioxygenase from W.satur- mus var.mrakii[4]andN. crassa [6]. However, NaoA activity is much higher with 1-nitropropane and nitro- ethane than with 2-nitropropane. We report some of the basic properties of NaoA. In the nitroalkane oxidation reaction, the superoxide anion is an essential intermediate [36]. On the basis of the inhibitory effects of various compounds on 2-nitropropane dioxyge- nase and by adding superoxide anion to the reaction mixture to induce nitroalkane oxygenation, it was demon- strated that superoxide anion indeed participates in the reaction as an intermediate [36], which is consistent with the reaction mechanism for 2-nitropropane denitrification to acetone proposed by Gorlatova et al.[6]andKuo& Fridovich [37]. In this study, NaoA activity was strongly inhibited by superoxide anion radical scavengers (nitro blue tetrazolium and NADH) as well as superoxide dismutase, which is in accord with the above 2-nitropropane dioxyg- enase [6,36], confirming that the superoxide anion is an essential intermediate in the nitroalkane oxidation catalyzed by NaoA. In addition, Gadda et al. [10,38] demonstrated that a cysteine residue and a neighboring tyrosine residue were present in the active site of the flavoprotein nitroalkane oxidase from F. oxysporum, whereas these two amino acids were not conserved in NaoA from S. ansochromogenes and the putative oxidoreductase from S. coelicolor. Possibly, NaoA uses another catalytic pathway. Mg 2+ and Ca 2+ did not affect NaoA activity, and are therefore probably not necessary for the oxidation. However, Cu 2+ strongly inhibited the activity of NaoA, and Mn 2+ slightly increased the activity. This implies that there may be a Mn 2+ or Cu 2+ binding site in the enzyme; metal ions may also act in other ways. We conclude that NaoA is a nitroalkane dioxygenase-like enzyme rather than a nitroalkane oxidase. Its characteristics are not identical with those of any reported nitroalkane- oxidizing enzymes, therefore it may be a novel enzyme able to convert nitroalkanes into the corresponding carbonyl compounds. 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However, Cu 2+ strongly inhibited the activity of NaoA, and Mn 2+ slightly. coli. Purification and characterization of NaoA Protein extracts of BL21(DE3)/pNA101 were separated by gel filtration, anion-exchange column chromatography, and ultrafiltration. The purified NaoA was further