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.
Kowlessur
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Laboratory of Neurochemistry, National Institute of Mental Health, National Institutes of Health, Building 36, Room 3D/30, 36, Convent Drive MSC 4096, Bethesda, MD 20892-4096, USA.
Kaufman
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D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D001933:Brain Stem / Q000201:enzymology; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D006836:Hydro-Lyases / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D010766:Phosphorylation; D010870:Pineal Gland / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D014365:Tryptophan Hydroxylase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001708:Biopterins; D014365:Tryptophan Hydroxylase; D006836:Hydro-Lyases; C076392:pterin-4a-carbinolamine dehydratase; C003402:sapropterin