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Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas several others have entered phases 2 and 3 of clinical trials. The present and future demand for pDNA requires the development of efficient bioprocesses to produce it. Commonly, pDNA is produced by cultures of Escherichia coli. It has been previously demonstrated that specific strains of E. coli with a modified substrate transport system can be able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. However, the large amounts of oxygen demanded can lead to microaerobic conditions after some hours of cultivation, even at small scale. Typically, the inherent problems for these cultures are the high oxygen demand and the accumulation of acetate, a metabolic byproduct that is synthesized aerobically when the glucose rate exceeds the limits.In recent years, several researches have been focused on the study of induction of plasmid DNA as well as strategies for fermentation using semi-defined mediums. These studies conceived relevant results that allow us to design a production platform for enhanced plasmid DNA. So, the main goal of this chapter is to show how the development of an experimental design directed to aromatic amino acids pathway can improve the yield of a therapeutic plasmid DNA by culture of a new strain of Escherichia coli VH33.

Fusariumgraminearum and related species commonly infest grains causing the devastating plant disease Fusarium head blight (FHB) and the formation of trichothecene mycotoxins. The most relevant toxin is deoxynivalenol (DON), which acts as a virulence factor of the pathogen. FHB is difficult to control and resistance to this disease is a polygenic trait, mainly mediated by the quantitative trait loci (QTL) Fhb1 and Qfhs.ifa-5A. In this study we established a targeted GC–MS based metabolomics workflow comprising a standardized experimental setup for growth, treatment and sampling of wheat ears and subsequent GC–MS analysis followed by data processing and evaluation of QC measures using tailored statistical and bioinformatics tools. This workflow was applied to wheat samples of six genotypes with varying levels of Fusarium resistance, treated with either DON or water, and harvested 0, 12, 24, 48 and 96 h after treatment. The results suggest that the primary carbohydrate metabolism and transport, the citric acid cycle and the primary nitrogen metabolism of wheat are clearly affected by DON treatment. Most importantly significantly elevated levels of amino acids and derived amines were observed. In particular, the concentrations of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan increased. No clear QTL specific difference in the response could be observed except a generally faster increase in shikimate pathway intermediates in genotypes containing Fhb1. The overall workflow proved to be feasible and facilitated to obtain a more comprehensive picture on the effect of DON on the central metabolism of wheat. Electronic supplementary material The online version of this article (doi:10.1007/s11306-014-0731-1) contains supplementary material, which is available to authorized users.

We had shown that aromatic amino acid (phenylalanine, tyrosine, and tryptophan) supplementation prevented bone loss in an aging C57BL/6 mice model. In vivo results from the markers of bone breakdown suggested an inhibition of osteoclastic activity or differentiation. To assess osteoclastic differentiation, we examined the effects of aromatic amino acids on early /structural markers as vitronectin receptor, calcitonin receptor, and carbonic anhydrase II as well as, late/functional differentiation markers; cathepsin K and matrix metalloproteinase 9 (MMP-9). Our data demonstrate that the aromatic amino acids down-regulated early and late osteoclastic differentiation markers as measured by real time PCR. Our data also suggest a link between the vitronectin receptor and the secreted cathepsin K that both showed consistent effects to the aromatic amino acid treatment. However, the non-attachment related proteins, calcitonin receptor, and carbonic anhydrase II, demonstrated less consistent effects in response to treatment. Our data are consistent with aromatic amino acids down-regulating osteoclastic differentiation by suppressing remodeling gene expression thus contributing initially to the net increase in bone mass seen in vivo.

The final publication is available at Springer via http://dx.doi.org/10.1007/s00344-013-9345-5 Quinate (1,3,4,5-tetrahydroxycyclohexanecarboxylate) is a compound synthesized in plants through a side branch of the shikimate biosynthesis pathway. Plants treated with herbicides that inhibit amino acid biosynthesis (branched-chain and aromatic) accumulate quinate in their leaves. The objective of this study was to evaluate whether quinate mimics the effects of herbicides in plants. In pea plants, exogenous application of quinate through the nutrient solution was compared with leaf spraying at a concentration of 4 and 400 mM, respectively, and evaluated in parallel to the effects of herbicides. The analysis facilitated an assessment of the phytotoxicity and potential use of quinate as a natural herbicide. The application of quinate through the nutrient solution, but not the spray, was lethal, although both treatments affected plant growth. Quinate was absorbed and translocated to other plant organs remote from the application site, and an increase in the levels of aromatic amino acids and caffeic acid (that is, compounds located after quinate in the shikimate biosynthesis pathway) was detected, which indicates that quinate was metabolized and incorporated into the shikimate pathway. Exogenous application of quinate affected the carbohydrate content in the leaves and roots in a way similar to the toxic effects of herbicides. The phytotoxic effects of quinate reported in this study suggest that this compound deregulates the shikimate pathway and mimics some physiological effects described in the mode of action of herbicides inhibiting amino acid biosynthesis. A. Zulet received a grant from the Spanish Ministry of Education and Science. This work was supported through funding from the Spanish Ministry of Science and Innovation (AGL- 2010-18621/AGR).

Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH) and nitric oxide synthase (NOS) are tetrahydrobiopterin (BH4)-dependent enzymes that catalyze the hydroxylation of the respective aromatic amino acids (PAH, TH and TPH) and the synthesis of NO from arginine (NOS), using dioxygen as additional substrate. While the aromatic amino acid hydroxylases all contain a catalytic mononuclear non-heme iron which is essential for the hydroxylation, NOS contains a cytochrome P450-type heme in the oxygenase domain where NO synthesis seems to take place. We have recently studied the structure of the complex of BH2 (the oxidized analogue of BH4) and substrate with PAH by NMR and docking 1 and, in order to get further insights on the role of the iron and tetrahydropterin cofactor in the catalytic mechanism in these enzymes, we have extended these studies to BH4. Based on the distance constraints obtained by NMR complemented by distance geometry calculations, docking into the crystal structure of the enzymes and molecular dynamic simulations, we have determined the conformation of BH4 bound to each of the four enzymes.

To examine the role of metallothionein (MT) on heavy metal accumulation in hepatic cytosol of rats, this study was carried out to determine the relative Zn, Cu, Cd, Au and Ag-binding capacities of MT in hepatic cytosol of Zn, Cu, Cd, Au and Aginjected rats, respectively. Our results demonstrated that approx. 60% of the Zn, Cu or Ag increments in the hepatic cytosol of Zn, Cu or Ag-injected rats was bound to MT, while approx. 80 or 4% of the Cd or Au increment in the hepatic cytosol of Cd or Au-injected rats was bound to MT. Therefore we suggested the order of the relative capacity in vivo of MT was determined for several metals (Cd>Zn=Ag≥Cu>Au). These results suggested that the role of MT in Zn, Ag or Cu accumulation in the liver of Zn, Ag or Cu-injected rats was different from that of MT in Cd or Au accumulation in the liver of Cd or Au-injected rats.

Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthetase (1, 2) as well as of aromatic amino acid hydroxylases of phenylalanine (3), tyrosine (4), and tryptophan (5, 6). BH4-supplement has been used effectively to treat many cases of neuronal dysfunctions caused by BH4 deficiency. Various life style-related diseases such as diabetes and hypertension are related to disorders in NO-synthesis and therefore they are potential targets for BH4-supplementation. Hence, BH4-supplements will be used in increasing numbers of these cases in the future. However, it is well known that BH4 administered orally is not taken up very efficiently. In our in vitro work using cultured RBL2H3 and PC-12 cells, uptake of BH4 was shown to be hampered by some cellular function to exclude it resulting in very inefficient incorporation (7), while sepiapterin (SP), a precursor of BH4 synthesis through the salvage pathway, was incorporated and converted to BH4 very efficiently (8). Further, the newly “synthesized” BH4 from exogenous SP was indistinguishable from endogenous BH4 in terms of its turnover rate and ability to support high tryptophan hydroxylase activity as a cofactor (9). Based on these observations, we searched for an effective method to increase tissue BH4 levels in normal mice. In this work, we observed that SP was efficiently taken up by mice and BH4 levels in various tissues increased much more and lasted longer than those resulting from 6RBH4 administration.

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, i.e., phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase, which enzymes catalyze the rate-limiting steps in the biosynthesis of neurotransmitters in the central nervous system. Sepiapterin reductase (SPR) is essential for BH4 biosynthesis by catalyzing the last step of the synthesis, and is important for controlling the level of BH4 in the brain. It has been reported that the BH4-requiring enzymes phenylalanine, tyrosine, and tryptophan hydroxylases are phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM KII). We have also found the phosphorylation of a BH4-recycling enzyme (dihydropteridine reductase) and SPR (sepiapterin reductase) by CaM KII in vitro (1). CaM KII, which is a Ser/Thr protein kinase, is known to occur abundantly in the brain, and is thought to play important roles in a variety of neuronal functions mediated by calcium ions (2). Recently, we reported that SPR phosphorylated by CaM KII was more sensitive to Ca2+-activated protease (calpain) than the non-phosphorylated form of SPR (3). This information suggests that the functions of both enzymes, i.e., those generating and those requiring BH4, may be regulated by CaM KII-dependent phosphorylation. In this study, we constructed various mutant SPRs and determined sites of SPR phosphorylated by CaM KII.

Tryptophan hydroxylase (TPH) is a tetrahydrobiopterin (BH4)- and iron-dependent enzyme that hydroxylates L-Trp to 5-hydroxy-L-Trp using molecular oxygen. This is the rate limiting step in the synthesis of serotonin. Due to the scarcity of the enzyme in animal tissues and its instability in vitro, TPH is the least characterized of the three aromatic amino acid hydroxylases and its 3D structure is still not known. Based on the high sequence identity between the three mammalian hydroxylases we have prepared a structural model for TPH (1) (Fig. 1A). We have also determined the structure of the bound conformation of L-Trp and the inactive cofactor analogue 7,8-dihydrobiopterin (BH2) complexed with a stable form of the catalytic domain of human TPH (Δ90TPH) by NMR and by molecular docking (McKinney et al., submitted; Fig. 1B). From the structure of the complex it was inferred that residue F313 (W326 in phenylalanine hydroxylase (PAH)) may have a role in substrate specificity. In this work we report the kinetic characterization of Δ90TPH and of the mutants F313/W326.

The tetrahydropterin-dependent aromatic amino acid hydroxylases phenylalanine hydroxylase (PheH) and tyrosine hydroxylase (TyrH) are 75% identical in their 335 C-terminal amino acid residues (1). Deletion mutagenesis and proteolysis of the native enzymes have shown that these amino acids contain the residues responsible for catalysis (2, 3, 4, 5, 6). Despite these extensive identities, these enzymes differ in their substrate specificities. PheH is very specific for phenylalanine; although it can hydroxylate tryptophan to a small extent, it is unable to hydroxylate tyrosine (7,8). TyrH is able to hydroxylate phenylalanine with approximately 66% the Vmax value with tyrosine (9,10). These substrate specificities are determined by the C domains (11). The goal of the experiments described here was to identify amino acid residues responsible for these different specificities.

In the familiar format of BNCT for cancer, the cells of a malignant tumor are sensitized to thermal neutrons by systemic administration of a B-10 containing compound that is selectively taken up by malignant cells. The boron-loaded tumor cells are then killed by neutron irradiation within a defined target volume. Effective BNCT requires a minimum of about 30μg 10B/g in the targeted malignant cells. To produce a useful therapeutic gain, there should be a differential of three or four fold between the boron concentration in malignant cells compared to that in the “normal” cells. This demanding pattern of boron delivery must be accomplished by a compound that has no serious toxicity to any vital tissue, either within the treatment volume or elsewhere in the body. Given these daunting requirements, it is remarkable that two compounds, BSH and L-Bpa, are currently in clinical trials, aspiring to the cure of glioblastoma multiforme. These gratifying results are hard won by many people over more than three decades. Hopefully, a careful choice of therapeutic targets, along with diligent attention to the literature of metabolite analog design, will allow new BNCT applications using new boron delivery molecules to reach clinical trial in a shorter time frame.

Prevailing boron carriers for BNCT are disodium mercaptoundecahydro-closo-dodecaborate (borocaptate sodium, BSH) and 4-dihydroxyboryl phenylalanine (4-boronophenylalanine, BPA).1,2 For clinical patient studies BPA is administred as an anionic fructose complex (BPA-F) infusions. As an aromatic amino acid without the fructose moiety BPA is a structural analogue for natural aromatic amino acid tyrosine. In BPA the hydroxyl group of tyrosine is substituted by the dihydroxyboryl group (or borono group), —B(OH)2. The dihydroxyboryl group is known to be fragile in aromatic molecules and B(OH)2-group can be substituted by electrophiles.3,4 However, by direct electrophilic fluorination of BPA with [18F]AcOF or [18F]F2 followed by HPLC separation, 4-borono-2-[18F]fluorophenylalanine (18FBPA) was prepared with radiochemical yields of 25-35% and with a radiochemical purity of over 99%.5 Clinical patient studies have shown that positron emission tomography (PET) with fluorinated analogue of BPA is a valuable technique for prediction of the effectiveness of boron neutron capture therapy using BPA as a boron carrier.6,7

A large number of plant products of commercial value including essential amino acids, alkaloids, phenols and structural compounds are derived from the shikimate pathway, but it is poorly understood what control architectures are required to produce these compounds. The creation of transgenic plants with artificial metabolic sinks that produce shikimate derived end-products or which effectively down-regulate the production of others has been successfully used in our laboratory to eliminate tryptophan-derived indoleglucosinolates inBrassica napus(canola). The elimination of indoleglucosinolates in canola seeds would be useful since this could increase the palatability of canola protein meals which are used for feeding swine and poultry. We have also shown that important effects on aromatic amino acid levels are observed when artificial metabolic sinks for tryptophan are introduced into transgenic plants. Transgenic potatoes which express tryptophan decarboxylase accumulate significantly lower levels of tryptophan and these plants were used to evaluate if tryptophan might play a role in the regulation of the shikimate pathway. Tubers from trans-genic potatoes also accumulated significantly decreased levels of phenylalanine, accumulated reduced levels of soluble and wall-bound phenolic compounds after wounding and were significantly more susceptible to infection byPhytopthora infestansa major fungal pathogen of potato. The significance of these results will be discussed in relation to the evolution of control architectures in microorganisms, fungi and plants which may regulate the shikimate pathway and which may result in different adaptive responses observed in plants when they accommodate a new artificial metabolic sink.

Naturally occurring pteridines in eukaryotic systems usually contain 2-amino, 4-hydroxy 6-alkyl substituents composed of either a methylene-p-aminobenzoylglutamate (or polutamate) or a dihydroxypropyl group. The former class are known collectively as the folates and occur widely as reduced and 5-alkylated derivatives, in which form they participate in important metabolic one-carbon transfers (Blakley, 1984). The latter, known as biopterin,, also occurs in reduced forms and is an important cofactor in aromatic amino acid hydroxylations en route to the catecholamines (Shiman, 1985; Kaufman and Kaufman, 1985; n and Lovenberg, 1985) and in the nitrite synthase pathway (Marletta,, 1993). Dihydrofolate reductase (DHFR), dihydropteridine reductase (DHPR) and pteridine reductase (PTR1), best characterised from Leishmania, are three enzymes that initiate the reduction of a pteridine in association with a reduced dinucleotide cofactor. Their comparative reaction pathways are illustrated in Figure 1.

Gallic acid (3,4,5-trihyroxybenzoic acid) serves as a basic building block of gallot annins and ellagitannins, which are abundant metabolites in plants and fungi. Tannins are produced by a wide variety of plants such as oak, acer, tea, and sumach (R. typhina). Several important functions such as stabilization of the cell wall and protection against herbivors have been attributed to tannins.1,2 More recently, certain tannins were reported to have antineoplastic activity, possibly via antioxidant activity.

Most aromatic compounds in plants are derived from shikimic acid metabolism; many of these substances are phenols. Compounds derived from this pathway are extensively modified and considered under other classes of plant secondary metabolites. Although many types of secondary compounds are produced from intermediates of the shikimic acid pathway (e.g., certain naphthoquinones and anthraquinones discussed in Chapter 6), most are derived from four aromatic amino acids: phenylalanine, tyrosine, anthranilic acid, and tryptophan. Aromatic compounds that arise from the shikimic acid pathway usually can be distinguished from those of other origins by their substitution patterns and by a knowledge of the compounds with which they co-occur.

Hepatic encephalopathy (HE) refers to a neuropsychiatric syndrome observed in patients with chronic liver failure. The pathogenesis for this important, but clinically often neglected, syndrome is still unknown. A profound perturbation in the handling of ammonia as well as of the essential aromatic amino acid L-tryptophan (L-TRP) has been shown to be associated with HE. This incapacity for normal L-TRP processing accompanying HE in vivo encompasses disturbances in L-TRP kinetics and metabolism both in brain tissue (1, for overview, see 2) as well as in the brain extracellular compartment (3,4).

Transport of branched-chain amino acids (BCAA) is of special importance for the metabolism of the brain. In contrast to other amino acids, there is net consumption of these amino acids by brain cells. The importance of this class of amino acids is underlined by the fact that the blood-brain barrier contains only one transport system for neutral amino acids, the well defined system L (1). The preferred substrates of this transport system are aromatic amino acids and BCAAs (2). Transport of neutral amino acids by system L is sodium independent and can be inhibited by the amino acid analogue 2-amino-bicyclo [2,2,l]heptane-2-carboxylic acid (BCH) (2). Two other transport systems for neutral amino acids are present in brain cells. These are the sodium dependent system A and the sodium dependent system ASC. Transport of neutral amino acids by system A can be inhibited by the amino acid analogue methylaminoisobutyric acid (MeAIB). Glial cells possess all three neutral amino acid transport systems (3, 4). However, BCAAs are poor substrates for system A and ASC because of the branched side chain. Transport of BCAAs is therefore mediated mainly by system L. At a concentration of 100 μM, 90% of isoleucine transport is mediated by system L. Two subtypes of this system are known, a high affinity system, designated L1 and a low affinity system, designated L2. In astrocytes and endothelial cells the L1 type is present (5). The activity of system L in astrocytes is very high (5, 6). Comparison with other transport systems in astrocytes shows that system L is one of the most active transport systems in glial cells. Release and uptake of BCAAs can be mediated by this system. It has been proposed that BCAAs and the corresponding 2-oxoacids constitute a pool of amino group donors and acceptors which can be rapidly distributed between the different cell types (6).

During the past few years, two novel types of redox cofactors have emerged, quinones and the free radical form of a specific amino acid residue in the enzyme protein chain. Quinone cofactors (Duine, 1991) (see Figure 1) can be either derived from tyrosine (PQQ, TPQ) or from tryptophan (TTQ). PQQ is non-covalently attached to the enzyme while TPQ and TTQ are protein-chain-integrated. TTQ consists of two tryptophans far apart in the protein chain and attached to each other via a covalent bond between their indole rings. It has sofar been discovered only in bacterial amine dehydrogenases (e.g. methylamine dehydrogenase, EC 1.4.99.3), enzymes involved in the oxidation of primary amines to the corresponding aldehydes and ammonia. Several amino acids have now been found which act as cofactor when oxidized to the free radical form in the protein chain. Most of them are derived from the aromatic amino acids tyr and trp and the cofactor TRP● (a tryptophyl residue in free radical form) has been discovered in several enzymes (Prince and George, 1990). The present status of this cofactor will be discussed.

Since Boron Neutron Capture Therapy is to be used in the treatment of primary and metastatic brain tumors, it is essential that the boron compounds be capable of crossing the blood-brain barrier (BBB) prior to their incorporation into tumor cells. It has been shown1 that derivatives of L-phenylalanine are transported across the BBB by neutral amino acid transport system. Recently, a method has been developed to deliver low molecular weight peptides into brain.2 These observations suggest that boronated analogues of phenylalanine itself and their peptides modified by replacing aromatic amino acids with highly lipophilic, carborane-containing amino acids might be used to reach and become incorporated into brain tumor cells protected by BBB.

Various drugs have been developed and trialed for boron neutron capture therapy (BNCT) but p-borono-phenylalanine (BPA) is one of the most successful compounds to date. BPA was originally developed as a selective targeting drug for BNCT of brain tumours on the basis that cancer cells require increased quantities of amino acids in order to maintain their faster metabolism compared to normal cells.1 Melanoma cells actively transport and metabolise aromatic amino acids such as phenylalanine and tyrosine for use as precursors in the synthesis of the pigment, melanin. BPA, being a phenylalanine derivative, is believed to exploit this mechanism resulting in the selective uptake in melanoma tumours and the successful application of BNCT in animals and some human patients.2–5

The aim of this work is to present information we gained on the structure and activity of human butyrylcholinesterase (BuChE) using high pressure techniques. Also, we would like to draw attention to the usefulness of the high pressure approach for understanding catalytic mechanisms and the molecular basis for structural stability of cholinesterases.

The amino acid sequences of several bacterial toxin ADP-ribosyltransferases, rabbit skeletal muscle transferases, and RT6.2, a rat T-cell NAD glycohydrolase, contain three separate regions of similarity, which can be aligned. Region I contains a critical histidine or arginine residue, region II, a group of closely spaced aromatic amino acids, and region III, an active-site glutamate which is at times seen as part of an acidic amino acid-rich sequence. In some of the bacterial ADP-ribosyltransferases, the nicotinamide moiety of NAD has been photo-crosslinked to this glutamate, consistent with its position in the active site. The similarities within these three regions, despite an absence of overall sequence similarity among the several transferases, are consistent with a common structure involved in NAD binding andADP-ribose transfer. (Mol Cell Biochem 138: 177–181, 1994)

The nucleocapsid protein NCp7 of the human immunodefiency virus type 1 (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C. NCp7 is thought to be a key component of the retrovirus life cycle since it activates Doth viral RNA dimerization and replication primer tRNALys,3 annealing to the initiation site of reverse transcription1,2. NCp7 constitutes thus a potential target for antiviral therapy.

The phenylalanine hydroxylating system is complex, consisting of three enzymes and two coenzymes1,2,3. One of the coenzymes, NADH, plays a near-ubiquitous role in intermediary metabolism. By contrast, the other cofactor, tetrahydrobiopterin (BH4), discovered during our early studies on the characterization of the phenylalanine hydroxylating system,1 plays a unique role as the essential coenzyme for certain hydroxylases such as the enzymes that catalyze the hydroxylation of the aromatic amino acids, phenylalanine, tyrosine and tryptophan3.

Tetrahydrobiopterin is a cofactor of the aromatic amino acid hydroxylases1. 6-Pyruvoyl tetrahydropterin(PTP) synthase is essential in the biosynthesis of tetrahydrobiopterin. This enzyme catalyzes the conversion of dihydroneopterin triphosphate to PTP in the presence of Mg2+2. The decrease of the amount of this enzymatic activity is the most frequent cause of atypical phenylketonuria3,4. PTP synthases were highly purified from human liver5, salmon liver6, and Drosophila head7. Recently, cDNA sequence of PTP synthase from rat liver was reported8. However, although PTP synthases were highly purified from several sources, the information on the enzymatic properties has been reported hardly anything.

Tetrahydrobiopterin (BH4) is the coenzyme for several monooxygenases such as the aromatic amino acid hydroxylases, the glycerol ether monooxygenase, and the nitric oxide synthases1,2. A lack of BH4 leads to hyperphenylalaninemia and a deficiency of biogenic amine neurotransmitters, which are responsible for severe mental retardation3. The most common form, where BH4 biosynthesis is impaired, is a deficiency in 6-pyruvoyl-tetrahydropterin synthase (PTPS). PTPS catalyzes the second step in the BH4 biosynthetic pathway, the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin. This triphosphate eliminating reaction requires Mg2+ as a cofactor. As a means to better characterize biochemically the PTPS, we recently cloned the human liver cDNA4. Expression of the recombinant enzyme in E. coli allowed us to isolate and purify the active PTPS in large amounts. This article describes the overproduction and purification, and gives a preliminary characterization of some physical properties of the recombinant human enzyme.

Dihydropteridine reductase (DHPR, EC 1.6.99.7) is the enzyme required for the recycling of tetrahydrobiopterin, an essential cofactor of the three aromatic amino acid hydroxylases (1). A rare inherited disorder is due to a deficiency of this enzyme, resulting in hyperphenylalanemia and disorders of dopamine and serotonin metabolism. DHPR-deficiency was recognized as an inheritable disorder, distinct from PKU, in the 1960’s, and the disease state was later correlated with an absence of enzyme activity in cultured fibroblasts (2). This lack of enzymatic activity was shown in some cases to be due to an absence of protein.

There is a paucity of detailed structural information on mammalian phenylalanine hydroxylase (PAH) mainly due to the fact that this enzyme has been difficult to crystallize. Equally there is a paucity of knowledge of the amino acids residues necessary for function. The main structural features described so far are shown in Table 1.

In many metabolic pathways a particular reaction step is catalyzed by different molecular forms of the same enzyme which are known as isoenzymes or simply isozymes. The isozymes are coded by different genes and differ in their amino acid composition. They may act as regulatory enzymes and control the metabolic fluxes and concentrations. An example is the metabolism of ethanol in horse liver by three isozymes of alcohol dehydrogenase1. Other examples include the branched biosynthetic pathway of the aromatic amino acids in E. coli. where the first enzyme, DAHP synthase, exists in the form of three isozymes each of which is inhibited by one of the three end products phenylalanine, tyrosine and tryptophan2.

(6R)-H4biopterin (BH4) is synthesized from GTP in different types of tissues and in cells of different lineages. It is the natural and immediate electron donor for aromatic amino acid monooxygenases. Thus, it serves as a cofactor in tissues committed to degradation of phenylalanine and the synthesis of the neurotransmitters dopamine, epinephrine, and serotonin (for review, see ref.1). The activity of GTP cyclohydrolase I (GTP-CH), the first and rate limiting enzyme of BH4 synthesis, appears to be constitutively expressed in all competent tissues such as liver, adrenal medulla, and brain2. Changes in its activity may occur in response to physiological conditions and pharmacological treatment3. No specific regulatory factors have been identified yet.

While enormous progress has been made in the elucidation of the molecular basis of amino acid transport in bacteria and lower eukaryotes, an understanding of the molecular principles that underlie mammalian amino acid transport is in its infancy. Investigations into the nature of amino acid transport in mammals has yielded a great wealth of knowledge on the substrate specificities and kinetic properties of amino acid transport systems. However, studies which seek to understand their molecular details have been complicated, to a large extent, by the relative low abundance of transport proteins in membranes, and the existence of different transport systems with overlapping specificities for individual amino acids. Several distinct transport systems mediate uptake of amino acids in mammalian cells. The major components for neutral amino acid uptake are systems A, ASC, and L which were first identified in Ehrlich ascites cells (Oxender and Christensen, 1963; Christensen et al., 1967). System A is sodium-dependent and serves predominantly for uptake of amino acids with short, polar or linear side chains. System L is sodium-independent, and shows reactivity toward branched-chain and aromatic amino acids. System ASC is sodium-dependent and shows a strong preference for alanine, serine, and cysteine. In this chapter we will outline and summarize the various approaches that have been used to gain insights into the molecular basis of the mammalian transport process. These approaches naturally divide into two broad areas of endeavor: (i) the identification and characterization of transport proteins, and (ii) the identification of transport genes and their mode(s) of regulating expression.

The plant aromatic pathway consists of three segments: the shikimate pathway segment that produces the aromatic amino acids phenylalanine, tyrosine, and tryptophan; the phenylpropanoid segment that produces the cinnamic acid derivatives that are precursors of flavonoids and the plant structural component lignin; and the flavonoid segment that produces the diverse flavonoid compounds. There is evidence that the three different sections should be treated as one metabolic unit and referred to as plant aromatic metabolism. Evidence further suggests that the aromatic metabolism is present early in plant development, at the stage when tissues differentiate.

The shikimic acid pathway1 has evolved in plants and microorganisms to provide for the synthesis of the aromatic amino acids, phenylalanine, tyrosine and tryptophan, as well as a number of other essential aromatic compounds, e.g., jo-aminobenzoic acid, the precursor of folic acid, or]D-hydroxybenzoic acid, the precursor of ubiquinone. A vast number of secondary metabolites, e.g., alkaloids or phenylpropanoids, are derived from these aromatic end products of the pathway. In addition, nature has invented a variety of diversions along the pathway which lead to a range of additional natural products. While the majority of secondary metabolites are derived from late segments of the shikimate pathway, at or beyond the stage of the branch point intermediate, chorismate, a few diversions also occur in the prechorismate part of the pathway. One of these, the reduction of shikimic acid to cyclohexanecarboxylic acid, forms the topic of this chapter.

The recent crystallographic analysis of a dimeric glycophospholipid form of AchE from Torpedo californica (Sussman et al., 1991) provides an ideal opportunity for comparing the catalytic behaviour and recognition properties of the enzyme with its atomic structure. The globular subunit is determined to be an α/s protein containing numerous crossover motifs of the type s-α-s or s-loop-s. The catalytic residue, Ser 200, resides 4 A above the base of a 20 A deep cavity - or gorge - lined with numerous aromatic amino acid residues accounting for approximately 40 percents of the active center surface. A catalytic charge-relay triad comprising Ser 200-His 440-Glu327 is identified. Quite surprisingly, only few net negative amino acid residues are found within the gorge: Asp 285 and Glu 273 at the top, Asp 72 about half-way into the gorge, and G1u 199, proximal to Ser 200, near the base. Overall, the atomic coordinates afford a picture of the active center as that of a deep, highly aromatic cavity, rich in pi-electron density. No evidence exists for an anionic choline-binding locus situated approximately 5 A from Ser 200.

One of the most notable features of strains of Acinetobacter is their ability to grow on a wide range of aromatic compounds. In addition, acinetobacters isolated from natural environments can grow in simple defined mineral media with no requirement for growth factors, so they are clearly synthesising all their own aromatic amino acids and related compounds. This review contains a description of the mechanisms used for the dissimilation of aromatic compounds by Acinetobacter strains; it concentrates on the best-characterised of these pathways, the mandelate pathway, but includes at least outline descriptions of the ways in which many other aromatic compounds are catabolised. In addition, there is a brief summary of the pathways for the biosynthesis of aromatic amino acids and an indication of the potential competition between these pathways and those for the degradation of aromatic compounds.

Green plants share with most microbial species the capacity to synthesize all the major amino acids; the few exceptions are parasitic and depend on the nitrogen metabolism of the host plant. In addition, many nonprotein “uncommon” amino acids are produced, and frequently accumulated, in large amounts in a range of species; these are important in plant/plant, plant/insect, and plant/animal relationships.1 The amino acids essential for mammalian diets are phenylalanine, tryptophan, lysine, leucine, isoleucine, valine, threonine, and methionine for adults, with the addition of arginine and histidine for immature animals (the third aromatic amino acid required for protein synthesis, tyrosine, can be formed metabolically, given an adequate dietary intake of phenylalanine). The biosynthetic routes for these 10 amino acids are particularly attractive targets for toxicologically safe herbicides and for pesticides aimed at plant pathogens; the potential for antibiotics may be more limited because medicinally important microorganisms tend to inhabit nutritionally rich habitats.

The shikimate pathway, a major route of carbon metabolism, leads to the biosynthesis of the three aromatic amino acids phenylalanine, tyrosine, and tryptophan (Herrmann, 1983; Pittard, 1987). In some plants, more than 20% of the fixed carbon flows through this pathway, the bulk for biosynthesis of secondary metabolites such as lignins, phytoalexins, and alkaloids.

Sickle hemoglobin under physiological conditions and concentrations aggregates upon de oxygenation to form a viscous gel composed of long fibers consisting of filaments of stacked rings, of which several detailed structures have been proposed (Finch et al., 1973). The important change in sickle hemoglobin is substitution of a non-polar hydrophobic residue (valine) for a polar residue (glutamic acid). This suggests that hydrophobic interactions are important in stabilizing HbS aggregation (Votano et al., 1977). A variety of small molecules containing hydrophobic moieties have been shown to inhibit the aggregation or gelation of deoxygenated HbS (Novak et al. 1978, 1979). These agents include aryalalkanes (Ross and Subramanian, 1977), the aromatic amino acids (Noguchi and Schechter, 1977, 1978), aliphatic alchols, amides and ureas (Poillon, 1980), a variety of oligopeptides (Kubota and Yang, 1977; Votano et al., 1977; Gorecki et al., 1980), and a variety of phenyl derivatives (Gorecki et al., 1980; Poillon, 1982). The inhibitory effects of these agents are believed to be due to a competitive interference mechanism in which the inhibitor binds to the HbS molecule at one of the important contact sites, and blocks other HbS molecules from binding at that site (Abraham et al., 1975; Behe and Englander, 1979).

Sepiapterin reductase [EC 1 .1.1.153](SPR) is an enzyme required in the biosynthesis of tetrahydrobiopterin (Katoh and Akino, 1986), an essential H-donor cofactor of aromatic amino acid hydroxylases such as tyrosine hydroxylase (Kaufman, 1986). Recently we found (Katoh and Sueoka, 1984; Sueoka and Katoh, 1985) that this enzyme belongs to the “Aldo-keto reductases” group (Tuner and Flynn, 1982; Wermuth, 1985). SPR can reduce various carbonyl compounds including “diketo-” compounds such as phenylpropanedione and diacetyl with NADPH (Katoh and Sueoka, 1984). The natural substrate of this enzyme, 6-pyruvoyl tetrahydropterin(6(R)-L-1′,2′-dioxopropyl 5,6,7,8-tetrahydropterin)(PPH4) is also a “diketo-” compound, and the vicinal “diketo” group (C1′-keto and C2′-keto) in the molecule was found to be reduced successively with NADPH by SPR to a “dihydrodiol” group to form tetrahydrobiopterin (BH4) (Masada et al., 1985: Curtius et al., 1985; Milstien and Kaufman, 1985; Brown et al., 1985; Smith and Nichol, 1986). In the previous works, only one type of mono-keto derivative (C2′-keto type) was found as the intermediate of SPR during the reduction of PPH4 at the usual assay pH (neutral pH). But recently we detected both types of the mono-keto derivatives (C1′-keto type and C2′-keto type) as the intermediate of the reaction if the reaction was performed at a pH lower than neutral (Katoh and Sueoka, 1990)(Fig. 1).

Metallothionein is a cadmium-binding protein first isolated from equine renal cortex by Margoshes and Vallee (1957). It is a protein of low molecular weight (about 6,000) having a very high cysteine content (about 30% of the amino acid residues) and an absence of aromatic amino acids and histidine (Kagi et al., 1974). Similar proteins have been isolated from the liver and/or kidney of humans (Pulido et al., 1966; Buhler and Kagi, 1974), and many other species. The amino acid sequences of equine renal metallothionein (Kojima et al., 1976) and hepatic metallothionein from mice (Huange et al., 1977) have been determined. The metallothionein from these two sources both contain 20 cysteine residues (out of a total of 61 amino acid residues) and remarkable structural homology in the amino acid sequence.

The last decade has witnessed a dramatic growth of interest in the administration of branched-chain amino acids as a possible therapy in patients with liver cirrhosis and hepatic encephalopathy. These patients show elevated levels of the aromatic amino acids (tyrosine, phenylalanine and tryptophan) as well as methionine, while the concentrations of the branched-chain amino acids (BCAA) leucine, isoleucine and valine are decreased.1,2,3 The aromatic amino acids are of particular interest since they serve as precursors for the physiological neurotransmitters norepinephrine, dopamine and serotonin. Moreover, they compete with the BCAA for transport across the blood-brain barrier via the same transport system, the L-system.4 As a result of the increased availability of aromatic amino acids, the reduced levels of BCAA and probably also an augmented permeability of the blood-brain barrier,5 the brain uptake of aromatic amino acids may increase. This in turn has been suggested to result in the formation of “false” neurotransmitters such as octopamine and phenylethanolamine.6 These amines are less biologically active than the physiological neurotransmitters and are thought to accumulate and displace the latter, thereby causing cerebral dysfunction.

Plants can synthesize everything they need to survive, including amino acids. The interrelationships of amino acid regulation and coordination with other metabolic processes has been studied for many years. One of the obstacles in this research has been the inability to probe individual pathways. Such obstacles preclude the understanding of the integration of these various pathways. An analogous situation existed for research on photosynthetic light reactions, and major advances in photosynthesis research were made with the discovery of potent, specific inhibitors of these pathways.

The conversion of L-tryptophan to L-5HTP is the first step in the biosynthesis of serotonin in the brain and of melatonin in the pineal gland and is catalyzed by tryptophan hydroxylase. Since the first demonstration by Grahame-Smith that a specific tryptophan hydroxylase exists in the brain (1), the enzyme has been studied at various laboratories including our own (2–11) and is now accepted as a pterin-dependent aromatic amino acid monooxygenase (12).

The mechanism of hepatic encephalopathy (HE) is still unknown. A characteristic amino acid pattern in HE has been described: an increased level of plasma free tryptophan, a fall in plasma branched chain amino acids (BCAA) valine, leucine and isoleucine and a rise in plasma aromatic amino acids (AAA) phenylalanine and tyrosine.1 The brain uptake of AAA would increase; the ensuing depletion of brain dopamine and norepinephrine and increase of brain octopamine, which is considered as a false neurotransmitter, would result in perturbation of cerebral neurotransmission.2