Expression and characterization of recombinant human and rat liver 6‐pyruvoyl tetrahydropterin synthase

Abstract

6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding -6-pyruvoyl-tetrahydropterin-synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent K, for the substrate dihydroneopterin triphosphate (8.5 pM for the human and 8.0 yM for the rat enzyme), PI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.Tetrahydrobiopterin ( B b ) is the cofactor of the aromatic amino acid hydroxylases [l] and other enzymes 12-41. Inherited 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency is the most frequent cause of an impaired metabolism of BH, [5]. The lack of BH, leads to hyperphenylalaninemia accompanied by severe progressive mental retardation [6]. Changes in BH, metabolism have also been observed in different neurological disorders such as Parkinson's disease, Alzheimer's disease, dystonia, depression and others [7-101. The committing step in the biosynthesis of BH, is the conversion of GTP to dihydroneopterin triphosphate (NH,TP) by GTP cyclohydrolase I [ll]. PTPS then converts NH,TP to 6-pyruvoyl tetrahydropterin, the first tetrahydro intermediate [12]. The last step in the BH, production is the

Mutation analysis of the 6‐pyruvoyl‐tetrahydropterin synthase gene in Chinese hyperphenylalaninemia caused by tetrahydrobiopterin synthesis deficiency

“…Therefore, the change of Asn52 to Ser52 (N52S) in human PTPS might destabilize the PTPS hexamer and cause the functional event. The proposed binding mode by Bürgisser et al (1994) suggested that the Glu107 and the amide bond between Thr105 and Thr106 (Glu108, Thr106, Thr107 in human) involved in the substrate anchoring by hydrogen bonding, and the Val69 (Val70 in human) lay close to the pocket of active site. The change of Thr106 and Val70 to Met106 (T106M) and Asp70 (V70D), respectively, found in PTPSdeficient patients might interfere with the substrate binding and cause a PTPS-deficient consequence.…”

Structural and Functional Consequences of Mutations in 6-Pyruvoyltetrahydropterin Synthase Causing Hyperphenylalaninemia in Humans

“…DNA Manipulations and Construction of Expression Vectors-All numbering of human PTPS cDNA and amino acid sequences refers to the published sequence (19). To obtain a recombinant wild-type PTPS lacking the N-terminal methionine, a pMal-c2 derivative was used as described before (14). Briefly, a DNA fragment was amplified by PCR from wild-type cDNA using primers PTPS101, containing a SalI site, and PTPS102, containing a BamHI site.…”

Structure of a 6-pyruvoyltetrahydropterin synthase homolog fromStreptomyces coelicolor

“…Site-directed mutagenesis studies have confirmed that the active site of PTPS has contributions from both toroids ( A , A ′ and B in Fig. 2), suggesting a hexameric biological unit (Bürgisser et al ., 1994, 1995; Ploom et al ., 1999). The trimeric PTPS homologs lack several key catalytic residues (Cys42, Asp88 and His89; see Fig.…”