Cloning and expression of recombinant human pineal tryptophan hydroxylase in Escherichia coli: purification and characterization of the cloned enzyme.

The first step in the biosynthesis of melatonin in the pineal gland is the hydroxylation of tryptophan to 5-hydroxytryptophan. A cDNA of human tryptophan hydroxylase (TPH) was cloned from a library of human pineal gland and expressed in Escherichia coli. This cDNA sequence is identical to the cDNA sequence published from the human carcinoid tissue 1]. This human pineal hydroxylase gene encodes a protein of 444 amino acids and a molecular mass of 51 kDa estimated for the purified enzyme. Tryptophan hydroxylase from human brainstem exhibits high sequence homology (93% identity) with the human pineal hydroxylase. The recombinant tryptophan hydroxylase exists in solution as tetramers. The expressed human pineal tryptophan hydroxylase has a specific activity of 600 nmol/min/mg when measured in the presence of tetrahydrobiopterin and L-tryptophan. The enzyme catalyzes the hydroxylation of tryptophan and phenylalanine at comparable rates. Phosphorylation of the hydroxylase by protein kinase A or calmodulin-dependent kinase II results in the incorporation of 1 mol of phosphate/mol of subunit, but this degree of phosphorylation leads to only a modest (30%) increase in BH(4)-dependent activity when assayed in the presence of 14-3-3. Rapid scanning ultraviolet spectroscopy has revealed the formation of the transient intermediate compound, 4alpha-hydroxytetrahydrobiopterin, during the hydroxylation of either tryptophan or phenylalanine catalyzed by the recombinant pineal TPH.