Tryptophan hydroxylase: cloning and expression of the rat brain enzyme in mammalian cells.

A cDNA encoding full-length tryptophan hydroxylase was produced by reverse transcriptase-PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated > 25-fold by Fe(II) and to a somewhat lesser extent by the polyanions heparin and phosphatidylserine. The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross-reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of approximately 220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme from brain.

Expression and deletion mutagenesis of tryptophan hydroxylase fusion proteins: delineation of the enzyme catalytic core.

cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1-98) or catalytic (amino acids 99-444) domains of the enzyme, were cloned and expressed as glutathione S-transferase fusion proteins in E. coli. The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S-transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity (Vmax) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99-444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.