Cloning and Determination of Kinetic Activity of Thermophilic GCH-I from Anoxybacillus flavithermus DSM 2641T

Yıl 2022, Cilt: 7 Sayı: 2, 178 - 183, 30.06.2022

Fatih Şaban Beriş , Özlem Hızal Hakan Karaoğlu

https://doi.org/10.35229/jaes.1068313

Öz

Phenylketonuria (PKU) is a disease caused by phenylalanine hydroxylase enzyme deficiency in newborn infants and is the most important cause of treatable mental retardation. One of the causes of the disease comes from the defects of the PTPS in the metabolic pathway of phenilalanine metabolisms. Treatment of the disease is not feasible, and life-time tetrahydrobiopterin loading is performed in chronic patients. Today, tetrahydrobiopterin is chemically synthesized. Biological production is a different point of view due to the long duration of chemical synthesis, costs, and exposure to chemical pollutants. For this reason, the gch-I gene from the thermophilic A. flavithermus DSM 2641T was identified by PCR method. We cloned the gchI gene that was 603 bp and its open reading frame has 200 amino acids. The gene was cloned into pET28a(+) expression vector with 6xHis tags and transform in E. coli BL21(DE3)pLys host cells to express with 1 mM IPTG induction. After purification with Ni-NTA resin, we determined that GCH-I is 24 kDa, its optimum pH is 8.0 and temperature is 65C.Under optimal conditions, GCH-I exhibited enzymatic activity with Km- and Vmax- values of 243 ± 23,25 μM and 100,93 ± 3,5 nM/min/mg protein, respectively.

Anahtar Kelimeler

A. flavithermus DSM 2641T, BH4, GCHI, PKU, Thermophilic bacterium

Destekleyen Kurum

Recep Tayyip Erdogan University Research Foundation

Proje Numarası

2011.102.03.4

Anoxybacillus flavithermus DSM 2641T Bakterisinin Termofilik GCH-I Enziminin Klonlanması ve Kinetik Aktivitesinin Belirlenmesi

Öz

Fenilketonüri (FKU) yenidoğan bebeklerde fenilalanin hidroksilaz enzim eksikliğinin neden olduğu bir hastalıktır ve tedavi edilebilir zihinsel geriliğin en önemli nedenidir. Hastalığın nedenlerinden biri, fenilalanin metabolizmasının metabolik yolundaki PTPS'nin kusurlarından kaynaklanmaktadır. Hastalığın tedavisi mümkün değildir ve kronik hastalarda yaşam boyu tetrahidrobiyopterin yüklemesi yapılır. Bugün, tetrahidrobiyopterin kimyasal olarak sentezlenir. Biyolojik üretim, uzun süre kimyasal sentez, maliyetler ve kimyasal kirleticilere maruz kalma nedeniyle farklı bir bakış açısıdır. Bu nedenle, termofilik A. flavithermus DSM 2641T'den gchI geni PCR yöntemi ile tanımlandı. 603 bp olan gchl genini klonlandı ve açık okuma çerçevesi 200 amino aside sahip olduğu tespit edildi. AfgchI geni, 6xHis etiketleri ile pET28a(+) ekspresyon vektörüne klonlandı ve 1 mM IPTG indüksiyonu ile eksprese etmek için E. coli BL21 (DE3)pLys konakçı hücrelerine transforme edildi. Ni-NTA afinite kromatografisi ile saflaştırıldıktan sonra GCH-I’in 24 kDa, optimum pH’sının 8,0 ve optimum sıcaklık isteminin 65C olduğu belirlendi. Optimal koşullar altında GCH-I, Km- ve Vmax- değerleri sırasıyla 243 ± 23,25 μM ve 100,93 ± 3,5 nM/dak/mg protein olarak tespit edildi.

Anahtar Kelimeler

A. flavithermus DSM 2641T, BH4, GCHI, FKU, Termofilik bakteri

Proje Numarası

2011.102.03.4

Kaynakça

• Andrade, C., Pereira, N. & Antranikian, G. (1999). Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes. Revista de Microbiologia, 30, 287-298.
• Blau, N. & Burgard, P. (2005). Disorders of phenylalanine and tetrahydrobiopterin metabolism. Physician's Guide to the Treatmentand Follow-up of Metabolic Diseases. N. Blau, G. Hoffmann, J. Leonard and J. Clarke. Heidelberg, Springer, pp 25-34.
• Blau, N., Thöny, B., Cotton, R.G.H. & Hyland, K. (2001). Disorders of tetrahydrobiopterin and related biogenic amines. In: Scriver, C. R., Beaudet, A. L., Sly, W. S., Vale, D., Childs, B., Vogelstein, B. (eds). The metabolic and molecular bases of inherited disease, 8th edn. McGraw-Hill, New York, pp 1725–1776.
• Bradford, M. (1976). A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72(1–2), 248–254.
• Celik, E. (2018). Termofilik Anoxybacillus flavithermus DSM2641T Bakterisinin ptps Geninin Klonlanması, Ekspresyonu ve Enzimin Biyokimyasal Karakterizasyonu, Yüksek Lisans Tezi, Recep Tayyip Erdoğan Üniversitesi Fen Bilimleri Enstitüsü. Rize, 57s.
• De Saizieu, A., Vankan, P. & van Loon, A. (1995). Enzymatic characterization of Bacillus subtilis GTP cyclohydrolase I. Biochem. J., 306, 371-377.
• El Yacoubi, B., Bonnett, S., Anderson, J.N., Swairjo, M.A., Iwata-Reuyl, D. & de Crecy-Lagard, V. (2006). Discovery of a New Prokaryotic Type I GTP Cyclohydrolase Family. The Journal of Biological Chemistry, 281(49), 37586–37593.
• Grochowski, L.L., Xu, H., Leung, K. & White, R.H. (2007). Characterization of an Fe(2+)-dependent archaeal-specific GTP cyclohydrolase, MptA, from Methanocaldococcus jannaschii. Biochemistry, 46, 6658-6667.
• Hartley, B.S., Hanlon, N., Jackson, R.J. & Rangrajan, M. (2000). Glucose isomerase: insight into protein engineering for increased thermostability. Biochem. Biophys. Acta, 1543, 294–335.
• Hızal, Ö. (2013). Anoxybacillus flavithermus Bakterisinin Isıl Kararlı Guanozintrifosfat Siklohidrolaz-I Geninin Klonlanması ve Ekspresyonu, Yüksek Lisans Tezi, Recep Tayyip Erdoğan Üniversitesi Fen Bilimleri Enstitüsü. Rize, 42s.
• Kalemci, M. (2020). Termofilik Anoxybacillus amylolyticus Bakterisinin gchI Geninin Klonlanması ve Enzimin Biyokimyasal Karakterizasyonu, Yüksek Lisans Tezi, Recep Tayyip Erdoğan Üniversitesi Fen Bilimleri Enstitüsü. Rize, 63s.
• Kure, S., Hou, D.C., Ohura, T., Iwamoto, H., Suzuki, S., Sugiyama, N., Sakamoto, O., Fujii, K., Matsubara, Y. & Narisawa, K. (1999). Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. The Journal of Pediatrics, 135(3), 375-378.
• Maita, N., Okada, K., Hatakeyama, K. & Hakoshima, T. (2002). Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP. PNAS, 99(3), 1212-1217.
• Moens, A.L. & Kass, D.A. (2006). Tetrahydropterin and Cardiovascular Disease. Arerioscler Thromb Vasc. Biol., 26, 2439-2444.
• Muntau, A.C., Röschinger, M., Habich, M., Demmelmair, H., Hoffmann, B., Sommerhoff, C.P. & Roscher, A.R. (2002). Tetrahydrobiopterin As An Alternative Treatment For Mild Phenylketonuria. N. Engl. J. Med., 347(26), 2122-2132.
• Özer, I. (2004). Fenilketonüri Örneğinde Doğumsal Metabolik Hastalıklarda Genel Tedavi Yaklaşımı. Klinik Pediatri, 3(1), 26-30.
• Ponzone, A., Spada, M., Ferraris, S., Dianzani, I. & Sanctis, L. (2004). Dihydropteridine Reductase Deficiency in Man: From Biology to Treatment. Medicinal Research Reviews, 24(2), 127-150.
• Saw, J.H., Mountain, B.W., Feng, L., Omelchenko, M.V., Hou, S., Saito, J.A., Stott, M.B., Li, D., Zhao, G., Wu, J., Galperin, M.Y., Koonin, E.V., Makarova, K.S., Wolf, Y. I., Rigden, D.J., Dunfield, P.F., Wang, L. & Alam, M. (2008). Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus WK1. Genome Biol., 9(11), R161.
• Spaaen, L. J. M., Bakker, J. A., Velter, C., Loots, W., Rubio-Gonzalbo, M.E., Forget, P.P., Dorland, L., De Koning, T.J., Poll-The, B.T., Ploos VaN Amstel, H.K., Bekhof, J., Blau, N. & Duran, M. (2001). Tetrahydropterin-responsive phenylalanine hydroxylase deficiency in Dutch neonates. J. Inherit. Metab. Dis., 24, 352-358.
• Suzuki, Y., Yasui, T. & Abe, S. (1979). Occurrence of GTP cyclohydrolase I in Bacillus stearothermophilus. J. Biochem., 86, 1679-1685.
• Tanaka, Y., Nakagawa, N., Kuramitsu, S., Yokoyama, S. & Masui, R. (2005). Novel reaction mechanism of GTP cyclohydrolase I. High-resolution X-ray crystallography of Thermus thermophilus HB8 enzyme complexed with a transition state analogue, the 8-oxoguanine derivative. J. Biochem., 138, 263-275.
• Taylor, I.N., Brown, R.C., Bycroft, M., King, G., Littlechild, J.A., Lloyd, M.C., Praquin, C., Toogood, H.S. & Taylor, S.J.C. (2004). Application of thermophilic enzymes in commercial biotransformation processes. Biochemical Society Transactions, 32(2), 290-292.
• Wang, H., Yang, B., Hao, G., Feng, Y., Chen, H., Feng, L., Zhao, J., Zhang, H., Chen, Y. Q., Wang, L., & Chen, W. (2011). Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina. Microbiology, 157, 3059–3070.
• Woo, H.J., Kang, J.Y., Choi, Y.K. & Park, Y.S. (2002). Production of Sepiapterin in Escherichia coli by Coexpression of Cyanobacterial GTP Cyclohydrolase I and Human 6-Pyruvoyltetrahydropterin Synthase. Applied and Environmental Microbiology, 68(6), 3138–3140.
• Yamamoto, K., Kataoka, E., Miyamoto, N., Furukawa, K., Ohsuye, K. &Yabuta, M. (2003). Genetic engineering of Escherichia coli for production of Tetrahydrobiopterin. Metabolic Engineering, 5, 246–254.
• Yim, J.J. & Brown, G.M. (1976). Characteristics of Guanosine Triphosphate Cyclohydrolase I Purified from Escherichia coli. The Journal of Biological Chemistry, 251(16), 5087-5094.
• Yoo, J.C., Han, J.M., Ko, O.H., & Bang, H.J. (1998). Purification and characterization of GTP cyclohydrolase I from Streptomyces tubercidicus, a producer of tubercidin. Arch. Pharm. Res. 21, 692-697.