TY - JOUR T1 - Evaluation of the Potential of Thermophilic Anoxybacteroides amylolyticum GCHI to Form BH4 Main-Substrate [*] TT - Termofilik Anoxybacteroides amylolyticum GCHI'nin BH4 Ana Substratını Oluşturma Potansiyelinin Değerlendirilmesi AU - Beriş, Fatih Şaban AU - Kalemci, Müride AU - Karaoğlu, Hakan AU - Aydın, Birsen Hacer PY - 2025 DA - November Y2 - 2025 DO - 10.35229/jaes.1808116 JF - Journal of Anatolian Environmental and Animal Sciences JO - JAES PB - Bülent VEREP WT - DergiPark SN - 2548-0006 SP - 1046 EP - 1052 VL - 10 IS - 6 LA - en AB - In this study, the cloning, expression, and biochemical characterization of GTP cyclohydrolase I, the first enzyme involved in phenylketonuria metabolism, from the thermophilic bacterium Anoxybacteroides amylolyticum was investigated. The gchI gene, identified by PCR from A. amylolyticum, is a single open reading frame encoding a total of 186 amino acids with a size of 555 bp. The expression of the enzyme, which catalyzes the conversion of GTP to dihydroneopterin triphosphate, was induced in E. coli BL21(DE3) cells using 1 mM IPTG cloned into pET28a(+). A Ni-NTA affinity column was used for purification of the enzyme. The characterization studies revealed that the enzyme's Vmax was 6.9x10-5 ± 2.3x10-4 μM/min/mg protein and its Km was 7.0 ± 1.4 mM. The enzyme showed the best activity at a temperature of 70°C and an optimal pH of 7.0. It was demonstrated that thermophilic A. amylolyticum GCHI could be used in the first step of the conversion of GTP under in vitro conditions as an alternative to the chemical synthesis of BH4, which is used in the treatment of phenylketonuria. KW - GTP KW - neopterin KW - GCHI KW - BH4 KW - Anoxybacteroides amylolyticum. N2 - Bu çalışmada, fenilketonüri metabolizmasında rol oynayan ilk enzim olan GTP siklohidrolaz I (GCHI)’in, termofilik bakteri Anoxybacteroides amylolyticum’dan klonlanması, ekspresyonu ve biyokimyasal karakterizasyonu araştırılmıştır. A. amylolyticum’dan PCR ile tanımlanan gchI geni, 555 bp boyutunda toplam 186 amino asidi kodlayan tek bir açık okuma çerçevesidir. GTP'nin dihidroneopterin trifosfata dönüşümünü katalize eden enzimin ekspresyonu, pET28a (+) içine klonlanarak 1 mM IPTG varlığında E. coli BL21(DE3) hücrelerinde gerçekleştirildi. Enzimin saflaştırılması için Ni-NTA afinite kolonu kullanıldı. Karakterizasyon çalışmaları enzimin, Vmax değerinin 6,9x10-5 ± 2,3x10-4 μM/dk/mg protein ve Km değerinin 7,0 ± 1,4 mM olduğunu ortaya koymuştur. Enzim, optimum 70°C sıcaklıkta 7,0 optimal pH’da en iyi aktiviteyi gösterdi. 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