Kegg Pathway: Tryptophan metabolism

Zhongguo Zhong Yao Za Zhi. 2008 Jul; 33(13): 1519-23
Sun SQ, Yan XF

OBJECTIVE: To investigate the effects of nitrogen forms on the camptothecin (CPT) content, Tryptophan synthase (TSB) and Tryptophan decarboxylase (TDC) activities in Camptotheca acuminata seedlings. METHOD: The seedlings of C. acuminata with 6 pairs of leaves were subjected to 5 different NH4(+) -N/NO3(-) -N ratio (0 : 100, 75 : 25, 50 : 50, 25 : 75, 100 : 0) treatments by sand culture in a greenhouse. The CPT content, TSB activity in the young leaves and TDC in the stem barks of the seedlings were determined by HPLC on the 15th, 30th, 45th, 60th and 75th day, respectively. RESULT: The obvious relationship between CPT content and nitrogen forms was observed. When NH4(+) - N /NO3(-) -N ratio was 25 : 75, CPT accumulation in young leaves displayed the best advantages (the highest value is 5.69 per thousand) and increased in the early 30 days of treatment and then declined. There was no obvious relationship between TSB activity in the young leaves and nitrogen forms. TDC activity in the stem bark was the highest when NH4(+) -N /NO3(-) -N ratio was 25 : 75, and the change of TDC activity paralleled to CPT content in the young leaves. CONCLUSION: A short-term treatment that NH4(+) -N /NO3(-) -N ratio was 25:75 may gain high CPT content in the young leaves through enhancing the TDC activity in the stem bark of C. acuminata seedlings.

Plant Physiol. 2008 Oct 1;
Malitsky S, Blum E, Less H, Venger I, Elbaz M, Morin S, Eshed Y, Aharoni A

In this study, transcriptomics and metabolomics data was integrated in order to examine the regulation of glucosinolate (GS) biosynthesis in Arabidopsis and its interface with pathways of primary metabolism. Our genetic material for analyses were transgenic plants overexpressing members of two clades of genes (ATR1-like and MYB28-like) that regulate the aliphatic and indole GSs biosynthetic pathways (AGs and IGs, respectively). We show that activity of these regulators is not restricted to the metabolic space surrounding GSs biosynthesis but is tightly linked to more distal metabolic networks of primary metabolism. This suggests that with similarity to the regulators we have investigated here, other factors controlling pathways of secondary metabolism might also control core pathways of central metabolism. The relatively broad view of transcript and metabolites altered in transgenic plants overexpressing the different factors underlined novel links of GSs metabolism to additional metabolic pathways including those of jasmonic acid, folate, benzoic acid and various phenylpropanoids. It also revealed transcriptional and metabolic hubs in the "distal" network of metabolic pathways supplying precursors to GSs biosynthesis and that over expression of the ATR1-like clade genes has a much broader effect on metabolism of indolic compounds than described previously. While the reciprocal, negative cross talk between the methionine and Tryptophan pathways that generate GSs in Arabidopsis has been suggested previously we now show that it is not restricted to AGs and IGs but includes additional metabolites, as for example the phytoalexin camalexin. Combining the profiling data of transgenic lines with gene expression correlation analysis allowed us to propose a model of how the balance in the metabolic network is maintained by the GSs biosynthesis regulators. It appears that ATR1/MYB34 is an important mediator between the two clade genes activity. While it is very similar to the ATR1-like clade members in terms of downstream gene targets, its expression is highly correlated with the one of the MYB28-like clade members. Finally, we used the unique transgenic plants obtained here to show that AGs are likely more potent deterrents of white fly as compared to IGs. The influence on insect behavior raises an important question for future investigation on the functional aspect of our initial finding that pointed to enriched expression of the MYB28-like clade genes in the abaxial domain of the Arabidopsis leaf.

metabolism. 2008 Oct; 57 Suppl 2: S6-10
Wurtman RJ

The brain is unusual among organs in that the rates of many of its characteristic enzymatic reactions are controlled by the local concentrations of their substrates, which also happen to be nutrients that cross the blood-brain barrier. Thus, for example, brain levels of Tryptophan, tyrosine, or choline can control the rates at which neurons synthesize serotonin, dopamine, or acetylcholine, respectively. The rates at which brain cells produce membrane phospholipids such as phosphatidylcholine (PC) are also under such control, both in adult animals and, especially, during early development. If pregnant rats are fed the 3 dietary constituents needed for PC synthesis- docosahexaenoic acid, uridine, and choline-starting 10 days before parturition and continuing for 20 days during nursing, brain levels of PC, and of the other membrane phosphatides (per cell or per mg protein), are increased by 50% or more. In adult animals, this treatment is also known to increase synaptic proteins (eg, synapsin-l, syntaxin-3, GluR-l, PSD-95) but not ubiquitous proteins like beta-tubulin and to increase (by 30% or more) the number of dendritic spines on hippocampal neurons. Docosahexaenoic acid currently is widely used, in human infants, to diminish the negative effects of prematurity on cognitive development. Moreover, docosahexaenoic acid, uridine (as uridine monophosphate), and choline are all found in mother's milk, and included in most infant formulas. It is proposed that these substances are part of a regulatory mechanism through which plasma composition influences brain development.

Hum Immunol. 2008 Sep 20;
Suzuki M, Konya C, Goronzy JJ, Weyand CM

Rheumatologists have long been focused on developing novel immunotherapeutic agents to manage such prototypic autoimmune disease as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The ultimate challenge in providing immunosuppressive treatment for patients with RA and SLE has derived from the dilemma that both protective and harmful immune responses result from adaptive immune responses, mediated by highly diverse, antigen-specific T and B cells endowed with powerful effector functions and the ability for long-lasting memory. As regulatory/suppressor T cells can suppress immunity against any antigen, including self-antigens, they emerge as an ideal therapeutic target. Several distinct subtypes of CD8(+) suppressor cells (Ts) have been described that could find application in treating RA or SLE. In a xenograft model of human synovium, CD8(+)CD28(-)CD56(+) T cells effectively suppressed rheumatoid inflammation. Underlying mechanisms involve conditioning of antigen presenting cells (APC). Adoptively transferred CD8(+) T cells characterized by IL-16 secretion have also exhibited disease-inhibitory effects. In mice with polyarthritis, CD8(+) Ts suppressed inflammation by IFNgamma-mediated modulation of the Tryptophan metabolism in APC. In SLE animal models, CD8(+) Ts induced by a synthetic peptide exerted suppressive activity mainly via the TGFbeta-Foxp3-PD1 pathway. CD8(+) Ts induced by histone peptides were found to downregulate disease activity by secreting TGFbeta. In essence, disease-specific approaches may be necessary to identify CD8(+) Ts optimally suited to treat immune dysfunctions in different autoimmune syndromes.

Neonatology. 2008 Sep 6; 95(2): 125-131
Hernandez-Rodriguez J, Meneses L, Herrera R, Manjarrez G

Background: The present study was aimed to obtain information on the interaction kinetics of L-Tryptophan (L-Trp) with plasma albumin from normal, intrauterine growth-restricted (IUGR) and nutritionally recovered (NR) newborn infants. Methods: A case study cohort was planned in 37 newborns during the first 3 months of life. At birth two groups were formed. The first group included 20 newborns with IUGR. The control group (C) included 17 appropriate for gestational age newborns. At 30 days of age, 9 infants of the IUGR group showed a return to normal of the anthropometric parameters, these infants formed the NR group. Free, bound and total L-Trp were measured. To assess binding kinetics albumin was freed of fatty acids and tested in mole to mole samples from IUGR, NR and control babies. Results: Plasma free L-Trp was increased, K(d) (dissociation constant) elevated and B(max )(maximal binding)decreased in IUGR patients up to postnatal day 90. These changes remained even after nutritional recovery. Conclusions: Abnormal kinetics of L-Trp binding to albumin explains the increased availability of this precursor amino acid in the plasma of IUGR infants. This finding corroborates previous results in IUGR rats and newborn babies, indicating enhanced potential for brain serotonergic synthesis.

Clin Chim Acta. 2008 Aug 8;
Lionetto L, Lostia AM, Stigliano A, Cardelli P, Simmaco M

BACKGROUND: The urinary excretion of vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) can be increased in the presence of neuroblastic and carcinoid tumors. The former is characterized by defective catecholamine metabolism which results in high urinary levels of VMA and HVA. The latter shows an altered metabolism of Tryptophan and an increased synthesis of serotonin, producing high 5-HIAA urinary concentrations. METHODS: We describe an HPLC-tandem mass spectrometric method for the simultaneous quantification of VMA, HVA and 5-HIAA in human urine. The chromatographic separation is performed on a reversed-phase C18 column. Instrumental analysis is performed on a Q-Trap 2000 triple quadrupole/ion trap mass spectrometer. RESULTS: The method is fast and does not require sample pre-treatment. Multiple calibration curve exhibited consistent linearity and reproducibility. Linear responses were observed in the concentration range 0-50 mg/l for each analyte. Limits of detection were 0.001 mg/l for VMA, 0.015 mg/l for 5-HIAA and 0.050 mg/l for HVA with a signal-to-noise ratio of 3. Limits of quantification were 0.005 mg/l for VMA, 0.050 mg/l for 5-HIAA and 0.1 mg/l for HVA with a signal-to-noise ratio of 10. CONCLUSIONS: This method can be proposed as a tool for neuroendocrine tumor markers detection.

Microbiology. 2008 Sep; 154(Pt 9): 2659-67
Chen H, Xu H, Kweon O, Chen S, Cerniglia CE

Enterococcus faecalis azoreductase (AzoA) is a very active enzyme with a broad spectrum of substrate specificity and is capable of degrading various azo dyes. The enzyme has an absolute requirement for reduced FMN, which delivers a total of four electrons from NADH to the substrate, resulting in the cleavage of the nitrogen double bond. In this study, we report the identification of amino acid residues critical for FMN binding in AzoA. FMN is stabilized by 22 amino acid residues, eight of which, Trp-105, Asn-106, Leu-107, Gly-150, Gly-151, Tyr-153, Asn-121 and Tyr-129, are involved in binding the FMN isoalloxazine ring. In silico analysis of the amino acid residues revealed that the Trp residue at position 105 of AzoA is the most likely significant contributor to the binding of FMN to the enzyme and is involved in FMN stabilization and destabilization. Site-directed mutagenesis analysis of Trp-105 was performed to determine the role of this amino acid residue in FMN binding and azo dye reductive activity. The mutant proteins were overexpressed in Escherichia coli and purified by anion-exchange and size-exclusion chromatography. The replacement of Trp-105 by the small side-chain amino acids Ala and Gly caused complete loss of both affinity for FMN and enzyme activity. Substitution of Tyr for Trp-105 did not significantly decrease the V(max) of the enzyme (22 % reduction). Substitutions with three bulky side-chain amino acids, Gln, Phe and His, produced enzymes with lower V(max) values (decreases of 68.2, 30.6 and 8.2-fold, respectively). However, these mutated enzymes maintained K(m) values similar to the wild-type enzyme. This study provides an insight into the catalytic properties of AzoA in FMN stabilization and enzyme activity.

Proc Natl Acad Sci U S A. 2008 Sep 9; 105(36): 13367-72
Wu Y, Kondrashkina E, Kayatekin C, Matthews CR, Bilsel O

The earliest kinetic folding events for (betaalpha)(8) barrels reflect the appearance of off-pathway intermediates. Continuous-flow microchannel mixing methods interfaced to small-angle x-ray scattering (SAXS), circular dichroism (CD), time-resolved Förster resonant energy transfer (trFRET), and time-resolved fluorescence anisotropy (trFLAN) have been used to directly monitor global and specific dimensional properties of the partially folded state in the microsecond time range for a representative (betaalpha)(8) barrel protein. Within 150 micros, the alpha-subunit of Trp synthase (alphaTS) experiences a global collapse and the partial formation of secondary structure. The time resolution of the folding reaction was enhanced with trFRET and trFLAN to show that, within 30 micros, a distinct and autonomous partially collapsed structure has already formed in the N-terminal and central regions but not in the C-terminal region. A distance distribution analysis of the trFRET data confirmed the presence of a heterogeneous ensemble that persists for several hundreds of microseconds. Ready access to locally folded, stable substructures may be a hallmark of repeat-module proteins and the source of early kinetic traps in these very common motifs. Their folding free-energy landscapes should be elaborated to capture this source of frustration.

J Bacteriol. 2008 Aug 29;
Ryu RJ, Patten CL

The plant growth-promoting rhizobacterium Enterobacter cloacae UW5 synthesizes the plant growth hormone indole-3-acetic acid (IAA) via the indole-3-pyruvate pathway utilizing the enzyme indole-3-pyruvate decarboxylase that is encoded by ipdC. In this bacterium, ipdC expression and IAA production occur in stationary phase and are induced by an exogenous source of Tryptophan, conditions that are present in the rhizosphere. The aim of this study was to identify the regulatory protein that controls expression of ipdC. We identified a sequence in the promoter region of ipdC that is highly similar to the recognition sequence for the E. coli regulatory protein TyrR that regulates genes involved in aromatic amino acid biosynthesis and metabolism. Using a tyrR insertional mutant, we demonstrated that TyrR is required for IAA production and for induction of ipdC transcription. TyrR directly induces ipdC expression as was determined by real-time qRT-PCR, by ipdC promoter-driven reporter gene activity, and by electrophoretic mobility shift assays. Expression increases in response to Tryptophan, phenylalanine, and tyrosine. This suggests that in addition to its function in plant growth-promotion, indolepyruvate decarboxylase is important for aromatic amino acid uptake and/or metabolism.

Cancer Res. 2008 Sep 1; 68(17): 7183-90
Neels OC, Koopmans KP, Jager PL, Vercauteren L, van Waarde A, Doorduin J, Timmer-Bosscha H, Brouwers AH, de Vries EG, Dierckx RA, Kema IP, Elsinga PH

[(11)C]-5-HydroxyTryptophan ([(11)C]HTP) and 6-[(18)F]fluoro-3,4-dihydroxy-l-phenylalanine ([(18)F]FDOPA) are used to image neuroendocrine tumors with positron emission tomography. The precise mechanism by which these tracers accumulate in tumor cells is unknown. We aimed to study tracer uptake via large amino acid transporters, peripheral decarboxylation (inhibited by carbidopa), and intracellular breakdown by monoamine oxidase (MAO). [(11)C]HTP and [(18)F]FDOPA tracer accumulation was assessed in a human neuroendocrine tumor cell line, BON. The carbidopa experiments were done in a tumor-bearing mouse model. Intracellular [(11)C]HTP accumulation was 2-fold higher than that of [(18)F]FDOPA. Cellular transport of both tracers was inhibited by amino-2-norbornanecarboxylic acid. The MAO inhibitors clorgyline and pargyline increased tracer accumulation in vitro. Carbidopa did not influence tracer accumulation in vitro but improved tumor imaging in vivo. Despite lower accumulation in vitro, visualization of [(18)F]FDOPA is superior to [(11)C]HTP in the neuroendocrine pancreatic tumor xenograft model. This could be a consequence of the serotonin saturation of BON cells in the in vivo model.

Biochemistry. 2008 Sep 23; 47(38): 10069-83
Chaves JM, Srivastava K, Gupta R, Srivastava OP

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic Tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of s