Evaluation of the Potential of Thermophilic Anoxybacteroides amylolyticum GCHI to Form BH4 Main-Substrate [*]

Authors : Fatih Şaban Beriş, Müride Kalemci, Hakan Karaoğlu, Birsen Hacer Aydın

Pages : 1046-1052

Doi:10.35229/jaes.1808116

View : 23 | Download : 102

Publication Date : 2025-11-30

Article Type : Research Paper

Abstract :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.
Keywords : GTP, neopterin, GCHI, BH4, Anoxybacteroides amylolyticum.

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