Mechanism of aromatic amino acid hydroxylation.
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ABSTRACT:
SUBMITTER: Fitzpatrick PF
PROVIDER: S-EPMC1635487 | biostudies-literature | 2003 Dec
REPOSITORIES: biostudies-literature
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Project description:The aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase are homotetramers, with each subunit containing a homologous catalytic domain and a divergent regulatory domain. The solution structure of the regulatory domain of tyrosine hydroxylase establishes that it contains a core ACT domain similar to that in phenylalanine hydroxylase. The isolated regulatory domain of tyrosine hydroxylase forms a stable dimer, while that of phenylalanine hydroxylase undergoes a monomer-dimer equilibrium, with phenylalanine stabilizing the dimer. These solution properties are consistent with the regulatory mechanisms of the two enzymes, in that phenylalanine hydroxylase is activated by phenylalanine binding to an allosteric site, while tyrosine hydroxylase is regulated by binding of catecholamines in the active site.
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Project description:The rare nonproteinogenic amino acid, meta-l-tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta-hydroxylation is unknown. Herein, we report the analysis of 3-hydroxylase (Phe3H) from Streptomyces coeruleorubidus. Insights from kinetic analyses of the wild-type enzyme and key mutants as well as of the biocatalytic conversion of synthetic isotopically labelled substrates and fluorinated substrate analogues advance understanding of the process by which meta-hydroxylation is mediated, revealing T202 to play an important role. In the case of the WT enzyme, a deuterium label at the 3-position is lost, whereas in in the T202A mutant 75 % retention is observed, with loss of stereospecificity. These data suggest that one of two possible mechanisms is at play; direct, enzyme-catalysed deprotonation following electrophilic aromatic substitution or stereospecific loss of one proton after a 1,2-hydride shift. Furthermore, our kinetic parameters for Phe3H show efficient regiospecific generation of meta-l-tyrosine from phenylalanine and demonstrate the enzyme's ability to regiospecifically hydroxylate unnatural fluorinated substrates.
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Project description:Fe (II)-and 2-ketoglutarate-dependent dioxygenases (Fe (II)/α-KG DOs) have been applied to catalyze hydroxylation of amino acids. However, the Fe (II)/α-KG DOs that have been developed and characterized are not sufficient. L-isoleucine dioxygenase (IDO) is an Fe (II)/α-KG DO that specifically catalyzes the formation of 4-hydroxyisoleucine (4-HIL) from L-isoleucine (L-Ile) and exhibits a substrate specificity toward L-aliphatic amino acids. To expand the substrate spectrum of IDO toward aromatic amino acids, in this study, we analyzed the regularity of the substrate spectrum of IDO using molecular dynamics (MD) simulation and found that the distance between Fe2+, C2 of α-KG and amino acid chain's C4 may be critical for regulating the substrate specificity of the enzyme. The mutation sites (Y143, S153 and R227) were also subjected to single point saturation mutations based on polarity pockets and residue free energy contributions. It was found that Y143D, Y143I and S153A mutants exhibited catalytic L-phenylalanine activity, while Y143I, S153A, S153Q and S153Y exhibited catalytic L-homophenylalanine activity. Consequently, this study extended the substrate spectrum of IDO with aromatic amino acids and enhanced its application property.
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Project description:To accurately detect and quantify low abundant transcripts no represented in current EST databases we generated an RNA-seq dataset based on RNA isolated from Petunia hybrida corolla tissue harvested at 8 PM at two developmental stages, day -1 (bud) and day 2 postanthesis (the tissues containing the lowest and highest levels of phenylalanine, respectively).
2015-09-09 | GSE70948 | GEO
Project description:To accurately detect and quantify low abundant transcripts no represented in current EST databases we generated an RNA-seq dataset based on RNA isolated from Petunia hybrida corolla tissue harvested at 8 PM at two developmental stages, day -1 (bud) and day 2 postanthesis (the tissues containing the lowest and highest levels of phenylalanine, respectively). Total RNA samples were obtained from corollas of at least eight wild-type petunia flowers per biological replicate at the two developmental stages using an RNeasy plant mini kit from Qiagen.
2015-09-09 | E-GEOD-70948 | biostudies-arrayexpress
Project description:Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.
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Project description:Human aromatic amino acid decarboxylase (AADC) is a pyridoxal 5'-phosphate-dependent enzyme responsible for the biosynthesis of dopamine and serotonin, essential neurotransmitters involved in motor and cognitive abilities. Mutations in its gene lead to AADC deficiency, a monogenic rare neurometabolic childhood parkinsonism characterized by severe motor and neurodevelopmental symptoms. Here, for the first time, we solved the crystal structure of human holoAADC in the internal aldimine (1.9 Å) and in the external aldimine (2.4 Å) of the substrate analog L-Dopa methylester. In this intermediate, the highly flexible AADC catalytic loop (CL) is captured in a closed state contacting all protein domains. In addition, each active site, composed by residues of both subunits, is connected to the other through weak interactions and a central cavity. By combining crystallographic analyses with all-atom and coarse-grained molecular dynamics simulations, SAXS investigations and limited proteolysis experiments, we realized that the functionally obligate homodimeric AADC enzyme in solution is an elongated, asymmetric molecule, where the fluctuations of the CL are coupled to flexibility at the edge between the N-terminal and C-terminal domains. The structural integrity of this peripheral protein region is essential to catalysis, as assessed by both artificial and 37 AADC deficiency pathogenic variants leading to the interpretation that structural dynamics in protein regions far from the active site is essential for CL flexibility and the acquirement of a correct catalytically competent structure. This could represent the molecular basis for pathogenicity prediction in AADC deficiency.
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Project description:Akebia trifoliata is a novel edible and healthy fruit. Here, we found that this fruit had the highest content of total free amino acids and three aromatic amino acids (AAAs) compared with the other popular fruits, and there was an obvious inverse relationship between AAA and flavonoid levels in various fruit tissues. Multiomics analysis revealed that the evolutionarily strengthened synthetic pathway of all three AAAs, the largely regulating ability conferred by ASP5 in the arogenate pathway and the complementary phenylpyruvate pathway endorsed by ADT of both Phe and Tyr biosynthesis provided reasonable explanations for the high AAA content in the flesh of A. trifoliata fruit. Gene-specific expression could be the main reason for the inverse relationship between AAAs and flavonoids. This study will help us understand the metabolic mechanism of AAAs and to develop A. trifoliata as a fresh fruit crop and medicinal plant by molecular breeding strategies.
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Project description:Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare inherited disorder that affects neurotransmitter biosynthesis. A DDC founder mutation c.714 + 4A > T (IVS6 + 4A > T) is prevalent in the Chinese population. This study investigated the epidemiology of AADC deficiency in Taiwan by analyzing data from National Taiwan University Hospital (NTUH), a central institution for diagnosing and treating the disease. From January 2000 to March 2023, 77 patients with AADC deficiency visited NTUH. Among them, eight were international patients seeking a second opinion, and another two had one or both non-Chinese parents; all others were ethnically Chinese. The c.714 + 4A > T mutation accounted for 85% of all mutated alleles, and 94% of patients exhibited a severe phenotype. Of the 77 patients, 31 received gene therapy at a mean age of 3.76 years (1.62-8.49) through clinical trials, and their current ages were significantly older than those of the remaining patients. Although the combined incidence of AADC deficiency in this study (1:66491 for 2004 and later) was lower than that reported in newborn screening (1:31997 to 1:42662), case surges coincided with the launch of clinical trials and the implementation of newborn screening. Currently, many young patients are awaiting for treatment.
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Project description:Organogels are semi-solid systems in which an organic liquid phase is immobilized by a 3-dimensional network composed of self-assembled gelator molecules. Although there is a large variety of organogel systems, relatively few have been investigated in the field of drug delivery, owing mostly to the lack of information on their biocompatibility and toxicity. In this work, organogelator-biocompatible structures based on aromatic amino acids, namely, tyrosine, tryptophan, and phenylalanine were synthesized by derivatization with aliphatic chains. Their ability to gel an injectable vegetable oil (i.e. safflower oil) and to sustain the release of a model anti-Alzheimer drug (i.e. rivastigmine) was then evaluated. Organogels and molecular packing were characterized by differential scanning calorimetry, rheology analysis, Fourier-transform infrared spectroscopy and X-ray crystallography. The amino acid derivatives were able to gel safflower oil through van der Waals interactions and H-bonds. Tyrosine-derivatives produced the strongest gels while tryptophan was associated with poor gelling properties. The superior gelling ability of tyrosine derivatives could be explained by their well-structured 2-dimensional packing in the network. The addition of an optimal N-methyl-2-pyrrolidone amount to tyrosine gels fluidized the network and allowed their injection through conventional needles. Upon contact with an aqueous medium, the gels formed in situ and released entrapped rivastigmine in a sustained fashion.
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