Role of Catalase and Superoxide Dismutase Activities on Oxidative Stress in the Brain of a Phenylketonuria Animal Model and the Effect of Lipoic Acid

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficiency of phenylalanine hydroxylase which leads to accumulation of phenylalanine and its metabolites in tissues of patients with severe neurological involvement. Recently, many studies in animal models or patients have reported the role of oxidative stress in PKU. In the present work we studied the effect of lipoic acid against oxidative stress in rat brain provoked by an animal model of hyperphenylalaninemia (HPA), induced by repetitive injections of phenylalanine and α-methylphenylalanine (a phenylalanine hydroxylase inhibitor) for 7 days, on some oxidative stress parameters. Lipoic acid prevented alterations on catalase (CAT) and superoxide dismutase (SOD), and the oxidative damage of lipids, proteins, and DNA observed in HPA rats. In addition, lipoic acid diminished reactive species generation compared to HPA group which was positively correlated to SOD/CAT ratio. We also observed that in vitro Phe inhibited CAT activity while phenyllactic and phenylacetic acids stimulated superoxide dismutase activity. These results demonstrate the efficacy of lipoic acid to prevent oxidative stress induced by HPA model in rats. The possible benefits of lipoic acid administration to PKU patients should be considered.     

Effect of glucagon on phenylalanine metabolism and phenylalanine-degrading enzymes in the rat

Glucagon administered subcutaneously to rats for 10 days had no significant effect on liver phenylalanine hydroxylase activity, but induced liver dihydropteridine reductase more than twofold. In rats administered a phenylalanine load orally, glucagon treatment stimulated oxidation and depressed urinary phenylalanine excretion. These responses could not be related to an effect of glucagon on hepatic tyrosine-alpha-oxoglutarate aminotransferase activity. Even in rats with phenylalanine hydroxylase activity depressed to 50% of control values by p-chlorophenylalanine administration, glucagon treatment increased the phenylalanine-oxidation rate substantially. Although hepatic phenylalanine-pyruvate aminotransferase was increased tenfold in glucagon-treated rats, glucagon treatment did not increase urinary excretion of phenylalanine transamination products by rats given a phenylalanine load. Glucagon treatment did not affect phenylalanine uptake by the gut or liver, or the liver content of phenylalanine hydroxylase cofactor. It is suggested that dihydropteridine reductase is the rate-limiting enzyme in phenylalanine degradation in the rat, and that glucagon may regulate the rate of oxidative phenylalanine metabolism in vivo by promoting indirectly the maintenance of the phenylalanine hydroxylase cofactor in its active, reduced state.     

A review of recent advances on the regulation of pigmentation in the human epidermis.

It has been recognised that the active transport of L-phenylalanine and its autocrine turnover to L-tyrosine via phenylalanine hydroxylase in the cytosol of epidermal melanocytes provides the majority of the L-tyrosine pool for melanogenesis. In this context, it has been shown that the cofactor 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) is produced de novo, recycled and regulated in both epidermal melanocytes and keratinocytes to control tyrosine hydroxylase, phenylalanine hydroxylase and tyrosinase activity. Inhibition of the enzymes by excessive 6BH4 levels is reversible with alpha-MSH by specific complex formation between 6BH4 and the hormone. This direct mechanism of alpha-MSH is supported by the presence of the entire POMC processing system in the melanosome indicating a receptor independent control of eumelanogenesis. Finally, the role of tyrosinase, TRP-1 and TRP2 is discussed in association with oxidative stress specifically related to hydrogen peroxide. These recent findings are based on detailed investigations of the depigmentation disorder vitiligo and Hermansky-Pudlák syndrome.     

Molecular Dynamics Simulations and Structure-Guided Mutagenesis Provide Insight into the Architecture of the Catalytic Core of the Ectoine Hydroxylase

Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.     

Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60°C and specific activity of about 2,600 U/mg with optimal temperature of 90°C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme''s activity.     

An additional substrate binding site in a bacterial phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum, a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site 15.7 Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 μM for phenylalanine. Under the same conditions, ITC revealed no detectable binding for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of amino acid residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) led to impaired binding, consistent with the presence of distal site binding in solution. Although kinetic analysis revealed that the distal site mutants suffer discernible loss of their catalytic activity, X-ray crystallographic analysis of Y155A and F258A, the two mutants with the most noticeable decrease in activity, revealed no discernible change in the structure of their active sites, suggesting that the effect of distal binding may result from protein dynamics in solution.     

Genetics of the mammalian phenylalanine hydroxylase system. IV. Evidence of phenylalanine hydroxylase in a cultured human hepatoma cell line

We report here the identification of a cultured human hepatoma cell line which possesses an active phenylalanine hydroxylase system. Phenylalanine hydroxylation was established by growth of cells in a tyrosine-free medium and by the ability of a cell-free extract to convert [¹⁴C]phenylalanine to [¹⁴C]tyrosine in an enzyme assay system. This enzyme activity was abolished by the presence in the assay system of p-chlorophenylalanine but no significant effect on the activity was observed with 3-iodotyrosine and 6-fluorotryptophan. Use of antisera against pure monkey or human liver phenylalanine hydroxylase has detected a cross-reacting material in this cell line which is antigenically identical to the human liver enzyme. Phenylalanine hydroxylase purified from this cell line by affinity chromatography revealed a multimeric molecular weight (estimated 275,000) and subunit molecular weights (estimated 50,000 and 49,000) which are similar to those of phenylalanine hydroxylase purified from a normal human liver. This cell line should be a useful tool for the study of the human phenylalanine hydroxylase system.     

Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.     

Tryptophan as a Molecular Shovel in the Glycosyl Transfer Activity of Trypanosoma cruzi Trans-sialidase

Molecular dynamics investigations into active site plasticity of Trypanosoma cruzi trans-sialidase, a protein implicated in Chagas disease, suggest that movement of the Trp³¹² loop plays an important role in the enzyme's sialic acid transfer mechanism. The observed Trp³¹² flexibility equates to a molecular shovel action, which leads to the expulsion of the donor aglycone leaving group from the catalytic site. These computational simulations provide detailed structural insights into sialyl transfer by the trans-sialidase and may aid the design of inhibitors effective against this neglected tropical disease.     

Large-scale conformational dynamics of the HIV-1 integrase core domain and its catalytic loop mutants

HIV-1 integrase is one of the three essential enzymes required for viral replication and has great potential as a novel target for anti-HIV drugs. Although tremendous efforts have been devoted to understanding this protein, the conformation of the catalytic core domain around the active site, particularly the catalytic loop overhanging the active site, is still not well characterized by experimental methods due to its high degree of flexibility. Recent studies have suggested that this conformational dynamics is directly correlated with enzymatic activity, but the details of this dynamics is not known. In this study, we conducted a series of extended-time molecular dynamics simulations and locally enhanced sampling simulations of the wild-type and three loop hinge mutants to investigate the conformational dynamics of the core domain. A combined total of >480 ns of simulation data was collected which allowed us to study the conformational changes that were not possible to observe in the previously reported short-time molecular dynamics simulations. Among the main findings are a major conformational change (>20 A) in the catalytic loop, which revealed a gatinglike dynamics, and a transient intraloop structure, which provided a rationale for the mutational effects of several residues on the loop including Q(148), P(145), and Y(143). Further, clustering analyses have identified seven major conformational states of the wild-type catalytic loop. Their implications for catalytic function and ligand interaction are discussed. The findings reported here provide a detailed view of the active site conformational dynamics and should be useful for structure-based inhibitor design for integrase.     

Immunochemical detection and characteristics of the subunit composition of phenylalanine hydroxylase in the brain of man

Immunochemical properties and subunit structure of an antigen were characterized in autopsy specimens of human liver and brain, using antiserum against human phenylalanine hydroxylase. An identical antigen was revealed in extracts of organs by immunoelectrophoresis. Its content was 1.5-2.0 mg/g tissue in the liver and 20-40 micrograms/g tissue in the brain. One L enzyme subunit and two H subunits were identified in the liver extracts after two-dimensional electrophoresis followed by immunoblotting. Subunit structure of phenylalanine hydroxylase in the brain was similar to that in the liver. The molecular weight of L subunit was 55,000 and it was located in the same area as albumin isoforms. The molecular weight of H subunits was 57,000 and they differed from L subunits in pI. The antigen was purified from crude extracts of biopsy liver by affinity chromatography on immunoadsorbent to phenylalanine hydroxylase and showed phenylalanine hydroxylase activity. An antigen with similar molecular weight was also purified from the brain extract by the same method. These data suggest that phenylalanine hydroxylase can be present in the human brain.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

Structural and stability effects of phosphorylation: Localized structural changes in phenylalanine hydroxylase

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.     

Long‐range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms

Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on six variants which differ from one another in terms of active and inactive form, with or without bound ligands, C‐terminal tail and phosphorylation at the activation loop. We find that the root mean square fluctuations in the MD simulations are generally higher for the inactive forms than the active forms. This difference is statistically significant. The higher dynamics of inactive states has significant contributions from ATP binding loop, catalytic loop, and αG helix. Simulations with and without C‐terminal tail show this differential dynamics as well, with lower dynamics both in the active and inactive forms if C‐terminal tail is present. Similarly, the dynamics associated with the inactive form is higher irrespective of the phosphorylation status of Thr 197. A relatively stable stature of active kinases may be better suited for binding of substrates and detachment of the product. Also, phosphoryl group transfer from ATP to the phosphosite on the substrate requires precise transient coordination of chemical entities from three different molecules, which may be facilitated by the higher stability of the active state.     

Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for L-DOPA formation

The catalytic domains of the pterin-dependent enzymes phenylalanine hydroxylase and tyrosine hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in tyrosine hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine hydroxylase identified three residues that contribute to the ability to hydroxylate tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine hydroxylase and the complementary mutations into tyrosine hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in tyrosine hydroxylase at the position corresponding to position 379 of phenylalanine hydroxylase is critical for dihydroxyphenylalanine formation. The relative specificity of tyrosine hydroxylase for phenylalanine versus tyrosine, as measured by the (V/K(phe))/(V/K(tyr)) value, increased by 80000-fold in the D425V enzyme. However, mutation of the corresponding valine 379 of phenylalanine hydroxylase to aspartate was not sufficient to allow phenylalanine hydroxylase to form dihydroxyphenylalanine at rates comparable to that of tyrosine hydroxylase. The double mutant V379D/H264Q PheH was the most active at tyrosine hydroxylation, showing a 3000-fold decrease in the (V/K(phe))/(V/K(tyr)) value.     

Structural investigation and inhibitory response of halide on phosphoserine aminotransferase from Trichomonas vaginalis

BackgroundPhosphoserine aminotransferase (PSAT) catalyses the second reversible step of the phosphoserine biosynthetic pathway in Trichomonas vaginalis, which is crucial for the synthesis of serine and cysteine.MethodsPSAT from T. vaginalis (TvPSAT) was analysed using X-ray crystallography, enzyme kinetics, and molecular dynamics simulations.ResultsThe crystal structure of TvPSAT was determined to 2.15 Å resolution, and is the first protozoan PSAT structure to be reported. The active site of TvPSAT structure was found to be in a closed conformation, and at the active site PLP formed an internal aldimine linkage to Lys 202. In TvPSAT, Val 340 near the active site while it is Arg in most other members of the PSAT family, might be responsible in closing the active site. Kinetic studies yielded Km values of 54 μM and 202 μM for TvPSAT with OPLS and AKG, respectively. Only iodine inhibited the TvPSAT activity while smaller halides could not inhibit.ConclusionResults from the structure, comparative molecular dynamics simulations, and the inhibition studies suggest that iodine is the only halide that can bind TvPSAT strongly and may thus inhibit the activity of TvPSAT.The long loop between β8 and α8 at the opening of the TvPSAT active site cleft compared to other PSATs, suggests that this loop may help control the access of substrates to the TvPSAT active site and thus influences the enzyme kinetics.General significanceOur structural and functional studies have improved our understanding of how PSAT helps this organism persists in the environment.     

Functional analysis of the effect of monoclonal antibodies on monkey liver phenylalanine hydroxylase.

An analysis of the effect of eleven monoclonal antibodies on the functional characteristics of monkey liver phenylalanine hydroxylase is presented. These eleven antibodies have been found to react with eight distinct regions on the phenylalanine hydroxylase protein. PH1 antibody inhibits enzyme activity, is dependent on phenylalanine for its binding, and appears to be related to structural changes occurring during phenylalanine activation of the enzyme activity. PH2 and PH3 antibodies stimulate enzyme activity, their binding is inhibited by lysolecithin and this group apparently is recognizing structures involved in lysolecithin activation of the enzyme activity. PH5, PH10, PH12 and PH6 recognise sites on phenylalanine hydroxylase affected by lysolecithin activation.     

Isolation of inactive phenylalanine hydroxylase from autopsy specimens of human liver

An electrophoretically homogeneous protein has been isolated from human liver autoptats, using a procedure employed for the isolation of phenylalanine hydroxylase from rat liver. The procedure includes chromatography of liver extracts on phenyl-Sepharose and subsequent purification on DEAE-Toyopearl. The activity of phenylalanine hydroxylase in the autoptats was markedly decreased in comparison with that in bioptats. The isolated protein possessed no enzymatic activity. However, the subunit composition of the protein, the molecular masses of protein subunits (55 and 57 kD) and the amino acid composition were close to those of the human enzyme. Antibodies to the protein inhibited the phenylalanine hydroxylase activity in human liver bioptats and weakly inhibited the rat enzyme. The experimental results suggest that the structural organization of phenylalanine hydroxylase does not alter as a result of the loss of enzymatic activity in cadaverous human liver.     

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.     

Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.