TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH Muhammad Atif Nisar,1 Naeem Rashid,1,* Qamar Bashir,1 Qurra-tul-Ann Afza Gardner,1 Muhammad Hassan Shafiq,1 and Muhammad Akhtar1,2 School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Punjab, Pakistan1 and School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK2 Received 27 November 2012; accepted 28 January 2013 Available online 26 February 2013 Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus koda- karaensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized. Molecular weight of the recombinant protein was 49,375 Da when determined by matrix-assisted laser desorption/ionization time-of-flight and 300 kDa when analyzed by gel filtration chromatography indicating that it existed in a hexameric form. The enzyme was highly thermostable even in boiling water where it exhibited more than 95% of the enzyme activity after incubation of 150 min. TK1299 catalyzed the oxidation of NADH as well as NADPH and predominantly converted O2 to H2O (more than 75%). Km value of the enzyme towards NADH and NADPH was almost same (24 ± 2 mM) where as specific activity was higher with NADPH compared to NADH. To our knowledge this is the most thermostable and unique NAD(P)H oxidase displaying higher enzyme activity with NADPH. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Hyperthermophile; Thermococcus kodakaraensis; NAD(P)H oxidase; Cloning; Circular dichroism; Thermostable] Nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a model hyperthermophilic organism whose whole genome has oxidases (NOXs, E.C.1.6.99.3) are members of “two dinucleotide been sequenced and reported (5). We have previously characterized binding domains” flavoproteins (tDBDF) superfamily. They catalyze two of the NADH oxidases, TK0304 (6) and TK1392 (7), out of seven the oxidation of NAD(P)H by reducing molecular oxygen to either present in T. kodakaraensis genome (8). In the present study we hydrogen peroxide (a two-electron reduction) or water (a four- focus on another NOX homolog, TK1299, from T. kodakaraensis. The electron reduction) or both (1). NOXs catalyze diverse redox reac- recombinant protein, produced in Escherichia coli, was purified and tions which are initiated by a stereospecific hydride transfer characterized in terms of its ability to reduce oxygen. between two cofactors, a pyridine nucleotide and a flavin adenine dinucleotide (FAD) (2). NOX homologs are widely distributed in MATERIALS AND METHODS members of all the three domains of life including eucarya, bacteria and archaea. Genomes analyses have revealed the presence of Bacterial strains, plasmids, and media E. coli DH5a cells were used for variety of NOX homologs in anaerobic archaea, a class of organisms cloning purposes and BL21-CodonPlus(DE3)-RIL (Stratagene, La Jolla, CA, USA) cells that have not been expected to possess NADH oxidases, indicating were employed for the expression of TK1299 gene that was cloned in pET-28a that they are well equipped for responding to oxidative stress. (Novagen, Madison, WI, USA). E. coli transformants were cultivated at 37 Cin e fi m Among anaerobic archaea the members of family Thermococcaceae Luria Bertani medium containing ampicillin at a nal concentration of 100 g/mL. contain multiple homologs of NOX (Table 1). Construction of the expression vector The structural gene, TK1299, fi Thermococcus kodakaraensis KOD1 is a hyperthermophilic encoding an NAD(P)H oxidase homolog was ampli ed by polymerase chain reaction (PCR) using genomic DNA of T. kodakaraensis as a template and a pair of primers archaeon isolated from a solfatara on the shore of Kodakara Island in consisting of TK1299-F (5-CCATGGAGAGAAAGACGGTCGTTG-3) and TK1299-R (5- Kagoshima, Japan (3,4). It is an obligate heterotroph, generally GTCAGAACTTGAGGACCCTGGC-3). Underlined sequence in TK1299-F indicates NcoI prefers proteinaceous matter as carbon and energy sources, and its recognition site. The amplified DNA fragment of 1.3 kbp was cloned in pTZ57R/T vector. The resulting plasmid was named pTZ-TK1299. TK1299 gene was liberated growth is associated with the reduction of elemental sulfur to H2S. It from pTZ-TK1299 using NcoI and BamHI restriction enzymes and cloned in pET-28a at the corresponding sites. The absence of any mutation in the cloned gene was confirmed by DNA sequencing. The resulting plasmid, pET-TK1299, was used to * Corresponding author. Tel.: þ92 42 99231534; fax: þ92 42 99230980. transform E. coli BL21-CodonPlus(DE3)-RIL for expression of TK1299. E-mail addresses: [email protected], [email protected] Expression and purification of recombinant TK1299 E. coli cells harboring (N. Rashid). pET-TK1299 were grown to early log phase and gene expression was induced for 4 h Reproduced from J. Biosci. Bioeng. 116: 39-44 (2013). Naeem Rashid: Participant of the 21st UM, 1993-1994. 353 TABLE 1. Distribution of NOX homologs in anaerobic archaea. temperatures ranging from 40Cto90C. Activation energy was calculated from straight line section of the Arrhenius plot. Thermodynamic parameters were Organism No. of homologs obtained by fitting the data to Eyring equation. Methanocaldococcus jannaschii 1 Circular dichroism analysis The structure stability of TK1299 was analyzed Methanococcus maripaludis 1 by circular dichroism (CD) spectroscopy in the presence or absence of the prosthetic Methanosarcina acetivorans 2 group (FAD). The protein samples were incubated at different temperatures ranging Methanosarcina barkeri 1 from 20Cto100C for 15 min in water bath or in autoclave for 5 min and CD spectra Pyrococcus abyssi 4 were taken. To examine the protein stability in the presence of cofactor, enzyme Pyrococcus furiosus 6 solutions were saturated with FAD and then incubated at different temperatures Pyrococcus horikoshii 5 before recording the CD spectra. Similarly the enzyme stability was also checked in Thermococcus gammatolerans 5 the presence of excess FAD. The CD spectra of the protein solutions were recorded in Thermococcus kodakaraensis 7 10 mM TriseCl pH 7.5 in the far UV-range of 200e260 nm. Solvent spectra were Thermococcus onnurineus 7 subtracted from those of the protein solutions. The spectra were recorded at Thermococcus sibiricus 5 Chirascan-plus CD Spectrometer (Applied Photophysics, UK). at 37 C using 0.4 mM isopropyl-thio-b-D-galactoside (IPTG) in LB medium con- RESULTS taining ampicillin at a final concentration of 100 mg/mL. The cells were harvested by  e centrifugation at 6000 g for 5 min at 4 C, resuspended in 50 mM Tris Cl (pH 8.0), Sequence analysis of TK1299 When multiple sequence and disrupted by sonication at 4C for 10 min. The supernatant after centrifugation was heated at 80C for 20 min. The heat labile proteins from the host cells were alignment of TK1299 was performed, using the amino acid denatured and removed by centrifugation at 13,000 Âg for 15 min at 4C. The sequence of its homologs, we could identify NAD(P)H binding heat-stable recombinant protein in the supernatant was further purified using motif, one coenzyme A disulfide reductase motif and two FAD fi ÄKTA Puri er chromatography system (GE Healthcare, Uppsala, Sweden). The binding motifs. Archaeal specific NADH oxidase amino acids (10) heat-treated cell extract was applied to anion-exchange Resource Q (6 mL) column (GE Healthcare) and recombinant TK1299 was eluted with a linear were also completely conserved in TK1299 (Fig. 1). TK1299 gradient of 0e1 M NaCl. The fractions containing TK1299 were dialyzed against displayed low homology (26e42%) to six NOX paralogs in 1.25 M (NH4)2SO4 in 50 mM sodium phosphate buffer (pH 7.0). The dialyzed T. kodakaraensis. enzyme sample was applied to hydrophobic Resource PHE (1 mL) column A phylogenetic tree was constructed by comparing the amino equilibrated with 1.25 M (NH4)2SO4 in 50 mM sodium phosphate buffer (pH 7.0) acid sequence of all the seven NADH oxidases found in and elution was done by decreasing the concentration of (NH4)2SO4. Analysis of the purified TK1299 was performed by sodium dodecyl sulfate polyacrylamide gel T. kodakaraensis genome and their homologs which resulted in electrophoresis (SDS-PAGE). seven discrete clusters (Fig. 2). Among the NADH oxidases that have Quantification of FAD bound to TK1299 To quantify FAD bound per mole of been characterized, TK1299 displayed highest homologies of 91% recombinant TK1299, 400 ml of TK1299 solution was mixed with 44 ml 50% tri- (identity) with that originated from Thermococcus profundus chloroacetic acid (TCA). The mixture was held on ice for 5 min and then centrifuged (accession no. CAQ43117) (11), 88 with Pyrococcus furiosus NAD(P)H at 12,000 rpm for 10 min to pellet down Apo-TK1299. The pellet was resuspended in elemental sulfur oxidoreductase (accession no. AAL81310) (12), 83% 400 ml 5% TCA and recentrifuged to recover an additional supernatant that was fi combined with that obtained from the first centrifugation. FAD in this combined to Pyrococcus horikoshii CoA disul de reductase (accession no. supernatant was quantified by measuring the absorbance at 450 nm. Calculations NP_142538) (13), and 60% with Archaeoglobus fulgidus NADH À À were based on absorption coefficient value of 11.3 mM 1 cm 1 for FAD (9). Apo- oxidase (accession no. NP_069231) (14). TK1299 displayed a low e TK1299, in the pellet, was dissolved in 50 mM Tris Cl pH 8.0 containing 0.2% homology with well-studied bacterial NOXs, 28% with NAD(P)H sodium dodecyl sulfate (buffer A). Spectra of apo-TK1299 were taken using buffer A as blank.