Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms.

Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed, purified and the kinetic properties have been studied and are compared. Substrate inhibition by tryptophan is observed for isoform 1 but not for isoform 2. Large differences are observed in the K (m,tetrahydrobiopterin) values for the two isoforms, being >10 times larger for isoform 1 compared to isoform 2.

Functional properties of missense variants of human tryptophan hydroxylase 2.

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the nervous system. Several variants of human TPH2 have been reported to be associated with a spectrum of neuropsychiatric disorders such as unipolar major depression, bipolar disorder, suicidality, and attention-deficit/hyperactivity disorder (ADHD). We used three different expression systems: rabbit reticulocyte lysate, Escherichia coli, and human embryonic kidney cells, to identify functional effects of all human TPH2 missense variants reported to date. The properties of mutants affecting the regulatory domain, that is, p.Leu36Val, p.Leu36Pro, p.Ser41Tyr, and p.Arg55Cys, were indistinguishable from the wild-type (WT). Moderate loss-of-function effects were observed for mutants in the catalytic and oligomerization domains, that is, p.Pro206Ser, p.Ala328Val, p.Arg441His, and p.Asp479Glu, which were manifested via stability and solubility effects, whereas p.Arg303Trp had severely reduced solubility and was completely inactive. All variants were tested as substrates for protein kinase A and were found to have similar phosphorylation stoichiometries. A standardized assay protocol as described here for activity and solubility screening should also be useful for determining properties of other TPH2 variants that will be discovered in the future.

Characterization of putative tryptophan monooxygenase from Ralstonia solanacearum [corrected].

The amino-acid sequence of a putative tryptophan monooxygenase (PTMO) from Ralstonia solanacearum is homologous with that of proenzyme (proPAO) of l-Phe oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 in their overall sequences. PTMO was expressed in E. coli and purified, but had no catalytic activity to oxidize l-Phe. By treating PTMO with various proteases, the Pronase-treated PTMO (PTMOp) showed a relatively high activity to oxidize l-Phe, l-Trp, l-Tyr and l-Met. Studies on the stoichiometry of the reaction showed that l-Phe and l-Tyr were mostly oxygenated, that l-Met was mostly oxidized, and both oxygenation and oxidation of l-Trp was observed. Initial velocity patterns were a ping-pong type with l-Phe and l-Tyr, and a sequential type with l-Trp and l-Met as substrate. The spectrum of enzymes with sufficient amounts of these substrates to reduce the enzyme showed a long wavelength species (purple complex) with l-Phe, but not with l-Tyr, l-Trp and l-Met. These results lead to the conclusion that PTMO and PTMOp belong to proPAO and PAO, respectively.

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization.

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.

Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris.

Tryptophan hydroxylase (TPH) from several mammalian species has previously been cloned and expressed in bacteria. However, due to the instability of wild type TPH, most successful attempts have been limited to the truncated forms of this enzyme. We have expressed full-length human TPH in large amounts in Escherichia coli and Pichia pastoris and purified the enzyme using new purification protocols. When expressed as a fusion protein in E. coli, the maltose-binding protein-TPH (MBP-TPH) fusion protein was more soluble than native TPH and the other fusion proteins and had a 3-fold higher specific activity than the His-Patch-thioredoxin-TPH and 6xHis-TPH fusion proteins. The purified MBP-TPH had a V(max) of 296 nmol/min/mg and a K(m) for L-tryptophan of 7.5+/-0.7 microM, compared to 18+/-5 microM for the partially purified enzyme from P. pastoris. To overcome the unfavorable properties of TPH, the stabilizing effect of different agents was investigated. Both tryptophan and glycerol had a stabilizing effect, whereas dithiothreitol, (6R)-5,6,7,8,-tetrahydrobiopterin, and Fe(2+) inactivated the enzyme. Irrespective of expression conditions, both native TPH expressed in bacteria or yeast, or TPH fusion proteins expressed in bacteria exhibited a strong tendency to aggregate and precipitate during purification, indicating that this is an intrinsic property of this enzyme. This supports previous observations that the enzyme in vivo may be stabilized by additional interactions.

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.

Advances in the molecular characterization of tryptophan hydroxylase.

The neurotransmitter serotonin has been implicated in numerous physiological functions and pathophysiological disorders. The hydroxylation of the aromatic amino acid tryptophan is rate-limiting in the synthesis of serotonin. Tryptophan hydroxylase (TPH), as the rate-limiting enzyme, determines the concentrations of serotonin in vivo. Relative serotonin concentrations are clearly important in neural transmission, but serotonin has also been reported to function as a local antioxidant. Identification of the mechanisms regulating TPH activity has been hindered by its low levels in tissues and the instability of the enzyme. Several TPH expression systems have been developed to circumvent these problems. In addition, eukaryotic expressions systems are currently being developed and represent a new avenue of research for identifying TPH regulatory mechanisms. Recombinant DNA technology has enabled the synthesis of TPH deletions, chimeras, and point mutations that have served as tools for identifying structural and functional domains within TPH. Notably, the experiments have proven long-held hypotheses that TPH is organized into N-terminal regulatory and C-terminal catalytic domains, that serine-58 is a site for PKA-mediated phosphorylation, and that a C-terminal leucine zipper is involved in formation of the tetrameric holoenzyme. Several new findings have also emerged regarding regulation of TPH activity by posttranslational phosphorylation, kinetic inhibition, and covalent modification. Inhibition of TPH by L-DOPA may have implications for depression in Parkinson's disease (PD) patients. In addition, TPH inactivation by nitric oxide may be involved in amphetamine-induced toxicity. These regulatory concepts, in conjunction with new systems for studying TPH activity, are the focus of this article.

Expression and characterization of the catalytic core of tryptophan hydroxylase.

Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4a-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.

Why tryptophan hydroxylase is difficult to purify: a reactive oxygen-derived species-mediated phenomenon that may be implicated in human pathology.

1. Attempts and apparently successful procedures to obtain reasonable quantities of electrophoretically homogenous mammalian brain-derived tryptophan hydroxylase, (TPH), have been described, starting in the early 1970s. This work has been carried out with the primary objective to obtain specific antisera to this enzyme to map out serotonergic pathways in the nervous system. 2. By using a multitude of techniques, antisera have indeed been fabricated and employed. However, it is doubtful if pure, native TPH has ever been produced. Indeed, there is strong evidence that more than one isoform of TPH exists in the rat brain. Thus, these antisera are probably directed against TPH-derived polypeptides and not the holoenzyme(s). 3. The difficulty in the purification of TPH lies not only in its subjectivity to proteolysis, but more importantly in its probable capacity to produce superoxide leading to hydrogen perioxide formation. This, in turn, may undergo Fenton chemistry with iron at the active site of the protein to produce hydroxyl radicals that directly attack and destroy the enzyme molecule. Evidence for such a mechanism is presented together with possible protocols that might be used to produce pure stable holo TPH(s). 4. It is hypothesized that similar oxidative events may take place in vivo under certain conditions leading to pathological results. Strategies to block these events are suggested.

Characterization of yellowfin tuna (Thunnus albacares, Scombroidei) tryptophan hydroxylase.

Tryptophan hydroxylase (EC 1.14, 16.4) was purified from yellowfin tuna liver and properties of this enzyme were compared with those of tryptophan hydroxylase from some other species (mouse mastocytoma and rat brain-stem). The molecular weight of the yellowfin tuna enzyme was estimated to be about 280,000 Da. This value is similar to that for the enzymes from mouse mastocytoma and rat brain-stem. On SDS-polyacrylamide gel electrophoresis analysis, yellowfin tuna enzyme was estimated to be about 96,000 Da. This value is different from that for the enzymes from mouse mastocytoma (53,000 Da) and rat brain-stem (59,000 Da) and suggests that yellowfin tuna enzyme may be a dimer of identical subunits of Mr 96,000 Da.

Tryptophan hydroxylase: purification by affinity chromatography on calmodulin-sepharose.

Tryptophan hydroxylase (EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)) from rat mesencephalic tegmentum has been purified by sequential chromatography on Blue-Sepharose, DE-52, and calmodulin-Sepharose. The hydroxylase is excluded from Blue-Sepharose and is eluted from DE-52 with a step-wise NaCl gradient. Tryptophan hydroxylase binds to calmodulin-Sepharose in the presence of calcium and is eluted with either EGTA or calmodulin itself, but not with tryptophan. The purification scheme is rapid (5-6 h) and yields an enzyme with a specific activity of 225 nmol 5-HTP/mg min, representing a 400-fold purification with 7% recovery. The tryptophan hydroxylase preparation was judged to be > 95% pure using the present isolation procedure.

High-level expression and deletion mutagenesis of human tryptophan hydroxylase.

Human tryptophan hydroxylase has been expressed as a soluble and active form in Escherichia coli by fusion with an affinity tag, maltose-binding protein. The fusion protein has been purified to near homogeneity by affinity chromatography on crosslinked amylose resin. The purified fusion protein has a specific activity of 86 nmol of 5-hydroxytryptophan per min per mg of fusion protein. A series of truncation mutants have also been made to explore the domain organization of tryptophan hydroxylase. All deletion mutants were subject to affinity purification and kinetic characterization. While removal of the N-terminal 164 amino acids completely inactivates the enzyme, deletion of the first 91 residues results in a 7-fold reduction in specific activity. From the C terminus, deletion of 36, 55, or 112 amino acids abolishes the activity, whereas deletion of 19 residues decreases the specific activity by approximately 11-fold. These results are consistent with a model for tryptophan hydroxylase in which the enzyme consists of an N-terminal regulatory domain, a catalytic core, and a small C-terminal region of uncertain but important function.

Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli.

Recombinant mouse tryptophan hydroxylase (TPH) was expressed in large quantities in Escherichia coli strain MC 1061, using a bacterial expression vector, pKS, containing the full coding region of mouse TPH. Specific polyclonal antiserum to the subunit of the recombinant mouse TPH was produced in rabbit by injecting the TPH band cut from SDS-polyacrylamide slab gels. The resultant antiserum recognized a single identical protein band (MW = 54,000) from rat dorsal raphe area, pineal gland, and brain stem by Western blot analysis. The specific activity of recombinant mouse TPH obtained was equivalent to that of TPH purified from rat brain. The recombinant mouse TPH was stable for 3 days at 4 degrees C but lost 25% of the original activity for the same period at -20 degrees C. A serotonin concentration greater than 1 mM inhibited TPH activity under our assay conditions in a concentration-dependent fashion. The recombinant mouse TPH exhibited a charge isozyme corresponding to that of pineal gland TPH as applied to chromatofocusing column chromatography. Taken together, our results show that recombinant mouse TPH, expressed in large quantities in E. coli is not only enzymatically highly active but also shares many biochemical and immunochemical properties with native TPH.

Inhibition of tryptophan hydroxylase by 6,7-dihydroxy-N-cyanomethyl-1,2,3,4-tetrahydroisoquinoline, a cyanomethyl derivative of dopamine formed from cigarette smoke.

6,7-Dihydroxy-N-cyanomethyl-1,2,3,4-tetrahydroisoquinoline, a cyanomethyl derivative of dopamine formed from cigarette smoke, was found to inhibit the activity of tryptophan hydroxylase. The inhibition was non-competitive to the substrate L-tryptophan (the Ki value 7.25 +/- 0.81 microM), but not to the biopterin cofactor. The inhibition is irreversible. 6-Hydroxy-N-cyanomethyl-tetrahydro-beta-carboline, a cyanomethyl derivative of serotonin, is inactive towards the hydroxylase. 6,7-Dihydroxy-N-cyanomethyl-1,2,3,4-tetrahydroisoquinoline may affect the serotonin biosynthesis in vivo as a consequence of cigarette smoking.

Tryptophan 5-hydroxylase. Rapid purification from whole rat brain and production of a specific antiserum.

Tryptophan 5-hydroxylase (EC 1.14.16.4; L-tryptophan tetrahydropteridine: oxygen oxidoreductase) was purified to electrophoretic homogeneity from whole brain supernatant using the following steps: pteridine-argarose affinity chromatography, hydrophobic and finally hydroxyapatite chromatography. Exogenous catalase was necessary throughout most of the purification procedure in order to protect the enzyme against inactivation. The iron chelator desferrioxamine at a concentration of 10 microM or higher brought about an irreversible loss of enzyme activity of a partially purified preparation containing an excess of catalase, whereas this same chelator at a lower concentration afforded considerable protection of the enzyme's activity during the final purification stage despite the quasi-total absence of catalase and the presence of an excess of ferrous iron. Antiserum raised in the rabbit to purified tryptophan 5-hydroxylase appears to be monospecific for the enzyme after immunoadsorption of anti-catalase antibodies which were present due to the trace of catalase which remained in the final enzyme preparation.

Regulation of 3-indoleacetic acid production in Pseudomonas syringae pv. savastanoi. Purification and properties of tryptophan 2-monooxygenase.

The oxidative decarboxylation of L-tryptophan to yield 3-indoleacetamide, catalyzed by tryptophan 2-monooxygenase, represents a controlling reaction in the synthesis of indoleacetic acid by Pseudomonas savastanoi (Pseudomonas syringae pv. savastanoi), a gall-forming pathogen of olive (Olea europea L.) and oleander (Nerium oleander L.). Production of indoleacetic acid is essential for virulence of the bacterium in its hosts. Tryptophan 2-monooxygenase was characterized to determine its role in indoleacetic acid metabolism in the bacterium. The enzyme was purified to apparent homogeneity from Escherichia coli cells containing the genetic locus for this enzyme obtained from P. savastanoi. The preparation contained a single polypeptide with a mass of 62,000 that cross-reacted immunologically with a homologous protein in P. savastanoi. The holoenzyme contained one FAD moiety/subunit with properties consistent with a catalytic function. The enzyme preparation catalyzed an L-tryptophan-dependent O2 uptake and yielded 3-indoleacetamide as a product. Enzyme activity fit simple Michaelis Menten kinetics with a Km for L-tryptophan of 50 microM. 3-Indoleacetamide and 3-indoleacetic acid were identified as regulatory effectors. The apparent Ki for 3-indoleacetamide was 7 microM; that for indoleacetic acid was 225 microM. At Km concentrations of tryptophan, enzyme activity was inhibited 50% by 25 microM 3-indoleacetamide. In contrast, 230 microM indoleacetic acid was required to effect a similar inhibition. Phenylalanine and tyrosine were ineffective as regulatory metabolites. These results indicate that IAA synthesis in P. savastanoi is regulated by limiting tryptophan and by feedback inhibition from indoleacetamide and indoleacetic acid.