View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 580 (2006) 4667–4672 Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K Shingo Kataokaa,b, Takatoshi Arakawaa, Shota Horia, Yoko Katayamac, Yoshiko Harac, Yasuhiko Matsushitad, Hiroshi Nakayamae, Masafumi Yohdaa, Hiroshi Nyunoyad, Naoshi Dohmaee, Mizuo Maedab, Masafumi Odakaa,b,* a Department of Biotechnology and Life Science, Graduate School of Technology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan b Maeda Bioengineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan c Department of Environmental and Natural Resource Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan d Gene Research Center, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan e Biomolecular Characterization Team, Advanced Development and Supporting Center, RIKEN, Wako, Saitama 351-0198, Japan Received 8 May 2006; revised 14 July 2006; accepted 14 July 2006 Available online 24 July 2006 Edited by Stuart Ferguson 1. Introduction Abstract Thiocyanate hydrolase (SCNase) is a cobalt-contain- ing enzyme with a post-translationally modified cysteine ligand, Thiocyanate is commonly produced from cellular thiogluco- cCys131-SO2H. When the SCNase a, b and c subunits were ex- pressed in Escherichia coli, the subunits assembled to form a het- sides in plant tissues [1] and exists as a major constituent of ero-dodecamer, (abc)4, like native SCNase but exhibited no waste water from coke oven factories. The degradation of thio- catalytic activity. Metal analysis indicated that SCNase was ex- cyanate is an important subject in green chemistry. Thiocya- pressed as an apo-form irrespective of the presence of cobalt nate-degradating organisms are believed to be distributed in the medium. On the contrary, SCNase co-expressed with widely. Thiocyanate hydrolase (SCNase) [2], catalyzing the P15K, encoded just downstream of SCNase genes, in cobalt-en- hydrolysis of thiocyanate to carbonyl sulfide and ammonia riched medium under the optimized condition (SCNase(+P15K)) (SCNÀ +2HO fi COS + NH +OHÀ), was first purified possessed 0.86 Co atom/abc trimer and exhibited 78% of the 2 3 from Thiobacillus thioparus THI 115. SCNase is composed of activity of native SCNase. SCNase(+P15K) showed a UV–Vis absorption peak characteristic of the SCNase cobalt center. a, b and c subunits. The SCNase c subunit is highly homolo- gous with the a subunit of nitrile hydratase (NHase), while the About 70% of SCNase(+P15K) had the cCys131-SO2H modifica- tion. These results indicate that SCNase(+P15K) is the active SCNase b and a subunits comprise the N- and C-terminal holo-SCNase. P15K is likely to promote the functional expres- halves of the NHase b subunit, respectively [3]. NHase [4] con- sion of SCNase probably by assisting the incorporation of cobalt tains a non-corrin cobalt or non-heme iron center, which is ion. bound to a motif, V-C1-X-L-C2-S-C3 of a subunit. C2 and Ó 2006 Federation of European Biochemical Societies. Published C3 are post-translationally modified to cysteine-sulfinic acid by Elsevier B.V. All rights reserved. (Cys-SO2H) and -sulfenic acid (Cys-SOH), respectively [5,6]. The motif is completely conserved in the SCNase c subunit, Keywords: Thiocyanate hydrolase; Nitrile hydratase; 127 133 Non-corrin cobalt; Activator protein; V -C-T-L-C-S-C [3]. Very recently, we have shown that Post-translational modification; SCNase possesses one Co ion per abc hetero-trimer with a Cysteine-sulfinic acid; Cysteine-sulfenic acid; Apo-protein similar ligand field with Co-type NHases [7]. We have also dis- covered that cCys131, corresponding to C2 in the motif, was post-translationally modified to Cys-SO2H based on mass spectrometric measurements [7]. Although the presence of the Cys-SOH modification has not been confirmed, these re- sults strongly suggest that SCNase belongs to the same protein superfamily as NHases. To characterize SCNase biochemically and structurally, we *Corresponding author. Address: Department of Biotechnology and tried to express SCNase in Escherichia coli. When only the Life Science, Graduate School of Technology, Tokyo University of genes coding SCNase a, b and c subunits were introduced into Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184- E. coli, no active SCNase was obtained even in the cobalt- 8588, Japan. Fax: +81 42 388 7479. enriched medium. It is known that both Co- and Fe-type E-mail address: [email protected] (M. Odaka). NHases require the co-expression of their specific accessory Abbreviations: SCNase, thiocyanate hydrolase; NHase, nitrile hydra- proteins [8–13]. We named them NHase activators [10]. tase; Cys-SO2H, cysteine sulfinic acid; Cys-SOH, cysteine sulfenic acid; Fe-type NHase activator has a putative metal-binding motif, IPTG, isopropyl-b-D-thiogalactopyranoside; SDS–PAGE, sodium CXCC, and the replacement of one of the three cysteines with dodecyl sulfate–polyacrylamide gel electrophoresis; SEC-MALS, size serine impaired functional expression of NHase in E. coli [14]. exclusion chromatography-multiangle laser light scattering; ESI-LC/ MS, electrospray ionization-liquid chromatography/mass spectro- In contrast, Co-type NHase activators are small proteins metry; CM, carboxy methylated with a molecular mass of 14–17 kDa and exhibit no sequence 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.07.051 4668 S. Kataoka et al. / FEBS Letters 580 (2006) 4667–4672 similarity to Fe-type ones [11–13,15,16]. It has no obvious ami- 26/60 (2.6 · 60 cm, Amersham Biosciences, USA), SuperQ-Toyopearl no acid sequence homology with known proteins except 650M (2.5 · 14 cm, Tosoh) and Butyl-Toyopearl 650S (2.5 · 20 cm, NHase b subunit and lacks known metal-binding motifs. Re- Tosoh). The purified SCNases were detected as a single band by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– cently, Cameron et al. [13] proposed that P14K, the Co-type PAGE). NHase activator of Bacillus pallidus RAPc8 functions as a sub- unit-specific chaperone. They speculated that P14K stabilized 2.3. Size exclusion chromatography-multiangle laser light scattering the structure of NHase a subunit to allow the incorporation of The molecular weight of apo-SCNase was determined using size Co ion prior to a-b subunit association. Here, we identified the exclusion chromatography-multiangle laser light scattering (SEC- SCNase activator, P15K, sharing only limited homology with MALS) on Shodex PROTEIN KW-804 (0.80 · 30 cm, Showadenko, Japan) and KW-803 (0.80 · 30 cm, Showadenko) columns connected Co-type NHase activators. Only when the SCNase a, b and c to a multiangle light-scattering detector (DAWN DSP, Wyatt Technol- subunits were expressed together with P15K in E. coli in ogy, USA) and a Shodex RI-71 differential refractive index detector cobalt-enriched culture medium, the obtained recombinant (Showadenko). Potassium phosphate (50 mM, pH 7.3) containing 100 mM KCl was used as the mobile phase at a flow rate of 1.0 mL/ SCNase exhibited SCNase activity. The results strongly 2 suggested that P15K promoted functional expression of SCN- min. 1.6 · 10 lg of apo-SCNase was injected into the column. The molecular weight and distribution of the recombinant protein were ase by assisting the incorporation of a cobalt ion into SCNase determined with the Program ASTRA [17] as described [18]. proteins. 2.4. Size exclusion chromatography The molecular weight and stoichiometry of the native and apo-SCN- 2. Materials and methods ases and were examined by SEC on Superdex 200 10/300 GL (1.0 · 30 cm, Amersham Biosciences) equilibrated with 50 mM potas- 2.1. Construction of expression plasmids sium phosphate, pH 7.8, containing 100 mM NaCl or with 50 mM To construct the plasmid for the expression of SCNase in E. coli, the potassium phosphate, pH 7.3, containing 100 mM KCl. The flow rate encoding sequence was amplified by PCR using a cosmid clone, SCN-2, was 0.50 mL/min. The amounts of the native and apo-SCNases in- containing scnB, scnA and scnC genes encoding the b, a and c sub- jected were 15 and 100 lg, respectively. The molecular weight and units of SCNase of T. thioparus THI 115 [3] as the template. The prim- stoichiometry of SCNase(+P15K) was examined by SEC on the same ers used were 50-TTTCATATGTCATCGTCCATCAGAGAAG-30 column equilibrated with 50 mM potassium phosphate, pH 7.8, con- and 50-TTTGAGCTCTCAATGATCGTGATGCAC-30. The PCR taining 100 mM NaCl. The flow rate was 0.50 mL/min. The amounts products were excised with NdeI and SacI and subcloned into pET32a of SCNase(+P15K) injected was 80 lg. (Novagen) to create the plasmid pGE32. To construct the plasmid for the expression of P15K in E. coli, the encoding sequence was amplified 0 2.5. Electrospray ionization-liquid chromatography/mass spectrometry by PCR using pUC118/T2 as the template. The primers used were 5 - Electrospray ionization-liquid chromatography/mass spectrometry AAGCATATGCCTGAGAACAACGTGGAAG-30 and 50-CCGAA- 0 (ESI-LC/MS) was performed with a Finnigan LCQ ion trap mass spec- GCTTTCAGACGCTCTTCAGTTTC-3 . The PCR products were trometer with an ESI probe, connected to a reversed-phase HPLC col- excised with NdeI and HindIII and subcloned into pET30a (Novagen) umn, Mightysil C8 (Kanto-kagaku, Japan) using an Agilent model to create the plasmid, pSAE30. As for the arabinose-induced expres- 1100 liquid chromatograph. The preparation of the peptide fragment sion, the expression vectors for SCNase subunits and P15K were con- containing the metal-binding motif and mass spectrometry were per- structed as follows: The scnB, scnA and scnC genes were amplified by formed as described [19].