Human Liver 6-Pyruvoyl-Tetrahydropterin Synthase: Expression of the cDNA, Purification and Preliminary Characterization of the Recombinant Protein

Abstract

Tetrahydrobiopterin (BH₄) is the coenzyme for several monooxygenases such as the aromatic amino acid hydroxylases, the glycerol ether monooxygenase, and the nitric oxide synthases^1,2. A lack of BH₄ leads to hyperphenylalaninemia and a deficiency of biogenic amine neurotransmitters, which are responsible for severe mental retardation³. The most common form, where BH₄ biosynthesis is impaired, is a deficiency in 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS catalyzes the second step in the BH₄ biosynthetic pathway, the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. This triphosphate eliminating reaction requires Mg²⁺ as a cofactor. As a means to better characterize biochemically the PTPS, we recently cloned the human liver cDNA⁴. Expression of the recombinant enzyme in E. coli allowed us to isolate and purify the active PTPS in large amounts. This article describes the overproduction and purification, and gives a preliminary characterization of some physical properties of the recombinant human enzyme.