Tyrosine, phenylalanine and tryptophan are the three aromatic amino acids (AAA) involved in protein synthesis. These amino acids and their metabolism are linked to the synthesis of a variety of secondary metabolites, a subset of which are involved in numerous anabolic pathways responsible for the synthesis of pigment compounds, plant hormones and biological polymers, to name a few. In addition, these metabolites derived from the AAA pathways mediate the transmission of nervous signals, quench reactive oxygen species in the brain, and are involved in the vast palette of animal coloration among others pathways. The AAA and metabolites derived from them also have integral roles in the health of both plants and animals. This review delineates the de novo biosynthesis of the AAA by microbes and plants, and the branching out of AAA metabolism into major secondary metabolic pathways in plants such as the phenylpropanoid pathway. Organisms that do not possess the enzymatic machinery for the de novo synthesis of AAA must obtain these primary metabolites from their diet. Therefore, the metabolism of AAA by the host animal and the resident microflora are important for the health of all animals. In addition, the AAA metabolite-mediated host-pathogen interactions in general, as well as potential beneficial and harmful AAA-derived compounds produced by gut bacteria are discussed. Apart from the AAA biosynthetic pathways in plants and microbes such as the shikimate pathway and the tryptophan pathway, this review also deals with AAA catabolism in plants, AAA degradation via the monoamine and kynurenine pathways in animals, and AAA catabolism via the 3-aryllactate and kynurenine pathways in animal-associated microbes. Emphasis will be placed on structural and functional aspects of several key AAA-related enzymes, such as shikimate synthase, chorismate mutase, anthranilate synthase, tryptophan synthase, tyrosine aminotransferase, dopachrome tautomerase, radical dehydratase, and type III CoA-transferase. The past development and current potential for interventions including the development of herbicides and antibiotics that target key enzymes in AAA-related pathways, as well as AAA-linked secondary metabolism leading to antimicrobials are also discussed.

Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phestabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.phenylalanine hydroxylase | phenylketonuria | X-ray crystallography | small-angle X-ray scattering | allosteric regulation M ammalian phenylalanine hydroxylase (PAH) (EC 1.14.16.1) is a multidomain homo-multimeric protein whose dysfunction causes the most common inborn error in amino acid metabolism, phenylketonuria (PKU), and milder forms of hyperphenylalaninemia (OMIM 261600) (1). PAH catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine, using nonheme iron and the cosubstrates tetrahydrobiopterin and molecular oxygen (2, 3). A detailed kinetic mechanism has recently been derived from elegant single-turnover studies (4). PAH activity must be carefully regulated, because although Phe is an essential amino acid, high Phe levels are neurotoxic. Thus, Phe allosterically activates PAH by binding to a regulatory domain. Phosphorylation at Ser16 potentiates the effects of Phe, with phosphorylated PAH achieving full activation at lower Phe concentrations than the unphosphorylated protein (5, 6). Allosteric activation by Phe is accompanied by a major conformational change, as evidenced by changes in protein fluorescence and proteolytic susceptibility, and by stabilization of a tetrameric confo...

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The first step of the shikimate pathway for aromatic amino acid biosynthesis is catalyzed by 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Thermotoga maritima DAH7PS (TmaDAH7PS) is tetrameric, with monomer units comprised of a core catalytic (␤/␣) 8 barrel and an N-terminal domain. This enzyme is inhibited strongly by tyrosine and to a lesser extent by the presence of phenylalanine. A truncated mutant of TmaDAH7PS lacking the N-terminal domain was catalytically more active and completely insensitive to tyrosine and phenylalanine, consistent with a role for this domain in allosteric inhibition. The structure of this protein was determined to 2.0 Å . In contrast to the wild-type enzyme, this enzyme is dimeric. Wild-type TmaDAH7PS was co-crystallized with tyrosine, and the structure of this complex was determined to a resolution of 2.35 Å . Tyrosine was found to bind at the interface between two regulatory N-terminal domains, formed from diagonally located monomers of the tetramer, revealing a major reorganization of the regulatory domain with respect to the barrel relative to unliganded enzyme. This significant conformational rearrangement observed in the crystal structures was also clearly evident from small angle X-ray scattering measurements recorded in the presence and absence of tyrosine. The closed conformation adopted by the protein on tyrosine binding impedes substrate entry into the neighboring barrel, revealing an unusual tyrosine-controlled gating mechanism for allosteric control of this enzyme.3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, 2 E.C. 2.5.1.54) catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) (Fig. 1). This reaction is the first step in the shikimate pathway, which is used to synthesize chorismate, the precursor of the aromatic amino acids phenylalanine, tyrosine, and tryptophan, and of other important aromatic metabolites (1). The shikimate pathway is found in plants and microorganisms, and it has more recently been shown to function in apicomplexan parasites (49). As the shikimate pathway is absent in animals, the enzymes of this pathway have been identified as possible targets for the development of antimicrobial agents (2). Regulation of the catalytic activity of DAH7PS has been shown to be an important mechanism for control of cellular levels of aromatic compounds in microorganisms and plants (3).13 C NMR studies using whole cells of Escherichia coli have demonstrated that feedback inhibition of DAH7PS is the main mechanism for controlling carbon flow into the shikimate pathway (4). Different organisms employ various strategies for this feedback inhibition. E. coli, Salmonella typhimurium, and Neurospora crassa express three DAH7PS isozymes, each sensitive to a single aromatic amino acid (5, 6). In Saccharomyces cerevisiae, there are two DAH7PS isozymes; one is inhibited by phenylalanine, and the other is inhibited by tyrosine (7). Other org...

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Neisseria meningitidis is the causative agent of meningitis and meningococcal septicemia is a major cause of disease worldwide, resulting in brain damage and hearing loss, and can be fatal in a large proportion of cases. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first reaction in the shikimate pathway leading to the biosynthesis of aromatic metabolites including the aromatic acids L-Trp, L-Phe, and L-Tyr. This pathway is absent in humans, meaning that enzymes of the pathway are considered as potential candidates for therapeutic intervention. As the entry point, feedback inhibition of DAH7PS by pathway end products is a key mechanism for the control of pathway flux. The structure of the single DAH7PS expressed by N. meningitidis was determined at 2.0 Å resolution. In contrast to the other DAH7PS enzymes, which are inhibited only by a single aromatic amino acid, the N. meningitidis DAH7PS was inhibited by all three aromatic amino acids, showing greatest sensitivity to L-Phe. An N. meningitidis enzyme variant, in which a single Ser residue at the bottom of the inhibitor-binding cavity was substituted to Gly, altered inhibitor specificity from L-Phe to L-Tyr. Comparison of the crystal structures of both unbound and Tyr-bound forms and the small angle X-ray scattering profiles reveal that N. meningtidis DAH7PS undergoes no significant conformational change on inhibitor binding. These observations are consistent with an allosteric response arising from changes in protein motion rather than conformation, and suggest ligands that modulate protein dynamics may be effective inhibitors of this enzyme.

Allosteric regulation of protein function is a critical component of metabolic control. Its importance is underpinned by the diversity of mechanisms and its presence in all three domains of life. The first enzyme of the aromatic amino acid biosynthesis, 3-deoxy-Darabino-heptulosonate 7-phosphate synthase, shows remarkable variation in allosteric response and machinery, and both contemporary regulated and unregulated orthologs have been described. To examine the molecular events by which allostery can evolve, we have generated a chimeric protein by joining the catalytic domain of an unregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with the regulatory domain of a regulated enzyme. We demonstrate that this simple gene fusion event on its own is sufficient to confer functional allostery to the unregulated enzyme. The fusion protein shares structural similarities with its regulated parent protein and undergoes an analogous major conformational change in response to the binding of allosteric effector tyrosine to the regulatory domain. These findings help delineate a remarkably facile mechanism for the evolution of modular allostery by domain recruitment.ACT domain | shikimate P rotein allostery, where ligand binding is coupled to a functional change at a remote site, is critical for the control of metabolism. For enzymes of key metabolic pathways, allostery allows precise control of catalysis in response to cellular demand. Although this important phenomenon was first described many years ago, it is only more recently that the molecular details that govern this precise control of enzyme function have been explored for a diverse range of protein systems (1-3). Functional change results from changes in the protein energy landscape elicited by ligand binding (4). This change in energy landscape may lead to conformational change and/or may be more subtly expressed as a change in protein molecular dynamics (5-12).The importance of protein allostery is underpinned by the observation that it is ubiquitous. As more proteins are studied in detail, one of the striking revelations is the variety of allosteric mechanisms that are used to control protein function. These mechanisms can be broadly divided into three groups that reflect the evolutionary path taken to acquire the allosteric functionality (5). In the first group, modification of existing structural features of a protein creates an allosteric site. Second, the formation of homo-and heterooligomeric assemblies may lead to the development of allosteric sites at the interface of subunits. Third, allostery may be endowed by domain fusion to create a modular protein with a distinct domain responsible for binding of the allosteric effector. A detailed understanding of this modular allostery, in which the allosteric effector binding site is associated with a discrete protein domain, offers the potential to generate engineered proteins with tailored functionality.The ACT domain has been identified as a modular regulatory unit associated with the control of a var...

Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-d--heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and displays a diverse range of allosteric mechanisms. DAH7PSs adopt a common architecture with a shared (β/α) catalytic domain which can be attached to an ACT-like or a chorismate mutase regulatory domain that operates via distinct mechanisms. These respective domains confer allosteric regulation by controlling DAH7PS function in response to ligand Tyr or prephenate. Starting with contemporary DAH7PS proteins, two protein chimeras were created, with interchanged regulatory domains. Both engineered proteins were catalytically active and delivered new functional allostery with switched ligand specificity and allosteric mechanisms delivered by their nonhomologous regulatory domains. This interchangeability of protein domains represents an efficient method not only to engineer allostery in multidomain proteins but to create a new bifunctional enzyme.