Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme

Abstract

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.

A typical N-terminal Extensions Confer Novel Regulatory Properties on GTP Cyclohydrolase Isoforms in Drosophila melanogaster

“…S2). Purified isoforms A-C consistently exhibit higher molecular masses on SDS-PAGE (39, 45, and 47 kDa, respectively) than the calculated molecular masses (30,34, and 36 kDa, respectively) as noted previously for Drosophila (38), rat (46), and human (47, 48) GTPCH subunits on SDS-PAGE. This discrepancy appears to be due, in part, to the N-terminal extensions of isoforms A-C, which exhibit apparent higher molecular masses (16,17, and 18 kDa, respectively) than expected (8,12, and 13 kDa, respectively), whereas the mobility of the common region is consistent with its expected molecular mass at 24 kDa.…”

“…In humans, there are at least six alternatively spliced GTPCH mRNAs, with the translated proteins differing only in their C termini, yet only GTPCH type I is enzymatically active (30). Co-expression of GTPCH type I and GTPCH type II, a truncated and non-functional GTPCH protein, in human blood cells depresses the level of GTPCH type I protein (31).…”

The eNOS cofactor tetrahydrobiopterin improves endothelial dysfunction in livers of rats with CCl4 cirrhosis

“…Only some of these isoforms have been shown to have enzymatic activity. 27 The primers used in the current study were designed to align with a zone supposed to be common in all mRNA species, suggesting therefore that we were amplifying all GTPCH mRNA species that exist in the liver.…”

Mutation in intron 5 of GTP cyclohydrolase 1 gene causes dopa-responsive dystonia (Segawa syndrome) in a Brazilian family

“…The GCH1 gene is constituted by 6 exons; it occupies approximately 30 kb in the genomic DNA and, at least six differential splicing variants differing on the 3' end have been described (Togari et al, 1992;Golderer et al, 2001;Hwu et al, 2003). However, only the full length transcript exhibits enzymatic activity (Gütlich et al, 1994). This transcript codes GTP cyclohydrolase I, an enzyme involved in the regulation of tetrahydrobiopterin (BH 4 ) synthesis, an essential co-factor for hydroxylases that converts phenylalanine, tryptophan, and tyrosine into tyrosine, serotonin and L-dopa, respectively (Müller et al, 2002).…”