Structure-function analyses of two plant meso-diaminopimelate decarboxylase isoforms reveal that active-site gating provides stereochemical control.

meso-Diaminopimelate decarboxylase catalyzes the decarboxylation of meso-diaminopimelate, the final reaction in the diaminopimelate l-lysine biosynthetic pathway. It is the only known pyridoxal-5-phosphate-dependent decarboxylase that catalyzes the removal of a carboxyl group from a d-stereocenter. Currently, only prokaryotic orthologs have been kinetically and structurally characterized. Here, using complementation and kinetic analyses of enzymes recombinantly expressed in Escherichia coli, we have functionally tested two putative eukaryotic meso-diaminopimelate decarboxylase isoforms from the plant species Arabidopsis thaliana We confirm they are both functional meso-diaminopimelate decarboxylases, although with lower activities than those previously reported for bacterial orthologs. We also report in-depth X-ray crystallographic structural analyses of each isoform at 1.9 and 2.4 Å resolution. We have captured the enzyme structure of one isoform in an asymmetric configuration, with one ligand-bound monomer and the other in an apo-form. Analytical ultracentrifugation and small-angle X-ray scattering solution studies reveal that A. thaliana meso-diaminopimelate decarboxylase adopts a homodimeric assembly. On the basis of our structural analyses, we suggest a mechanism whereby molecular interactions within the active site transduce conformational changes to the active-site loop. These conformational differences are likely to influence catalytic activity in a way that could allow for d-stereocenter selectivity of the substrate meso-diaminopimelate to facilitate the synthesis of l-lysine. In summary, the A. thaliana gene loci At3g14390 and At5g11880 encode functional. meso-diaminopimelate decarboxylase enzymes whose structures provide clues to the stereochemical control of the decarboxylation reaction catalyzed by these eukaryotic proteins.

A Three-Ring Circus: Metabolism of the Three Proteogenic Aromatic Amino Acids and Their Role in the Health of Plants and Animals.

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.

Exploring modular allostery via interchangeable regulatory domains.

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-arabino-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 (ß/α)8 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.

The Functional Unit of Neisseria meningitidis 3-Deoxy-á´ -Arabino-Heptulosonate 7-Phosphate Synthase Is Dimeric.

Neisseria meningitidis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimeric DAH7PS in solution, as determined by small angle X-ray scattering, analytical ultracentrifugation and analytical size-exclusion chromatography. The dimeric NmeDAH7PSR126S variant was shown to be catalytically active in the aldol-like condensation reaction between D-erythrose 4-phosphate and phosphoenolpyruvate, and allosterically inhibited by L-phenylalanine to the same extent as the wild-type enzyme. The dimeric NmeDAH7PSR126S variant exhibited a slight reduction in thermal stability by differential scanning calorimetry experiments and a slow loss of activity over time compared to the wild-type enzyme. Although NmeDAH7PSR126S crystallised as a tetramer, like the wild-type enzyme, structural asymmetry at the less extensive interface was observed consistent with its destabilisation. The tetrameric association enabled by this Arg126-Glu27 salt-bridge appears to contribute solely to the stability of the protein, ultimately revealing that the functional unit of NmeDAH7PS is dimeric.

First structure of full-length mammalian phenylalanine hydroxylase reveals the architecture of an autoinhibited tetramer.

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 Phe-stabilized 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.

Allosteric ACTion: the varied ACT domains regulating enzymes of amino-acid metabolism.

Allosteric regulation of enzyme activity plays important metabolic roles. Here we review the allostery of enzymes of amino-acid metabolism conferred by a discrete domain known as the ACT domain. This domain of 60-70 residues has a ßαßßαß topology leading to a four-stranded ß4ß1ß3ß2 antiparallel sheet with two antiparallel helices on one face. Extensive sequence variation requires a combined sequence/structure/function analysis for identification of the ACT domain. Common features include highly varied modes of self-association of ACT domains, ligand binding at domain interfaces, and transmittal of allosteric signals through conformational changes and/or the manipulation of quaternary equilibria. A recent example illustrates the relatively facile adoption of this versatile module of allostery by gene fusion.

Structural analysis of substrate-mimicking inhibitors in complex with Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase - The importance of accommodating the active site water.

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholactate, (S)-phospholactate and vinyl phosphonate [(E)-2-methyl-3-phosphonoacrylate], were found to competitively inhibit DAH7PS from Neisseria meningitidis, which is the pathogen responsible for bacterial meningitis. The most potent inhibitor was the vinyl phosphonate with a Ki value of 3.9±0.4µM. We report for the first time crystal structures of these compounds bound in the active site of a DAH7PS enzyme which reveals that the inhibitors bind to the active site of the enzyme in binding modes that mimic those of the predicted oxocarbenium and tetrahedral intermediates of the enzyme-catalysed reaction. Furthermore, the inhibitors accommodate the binding of a key active site water molecule. Together, these observations provide strong evidence that this active site water participates directly in the DAH7PS reaction, enabling the facial selectivity of the enzyme-catalysed reaction sequence to be delineated.

Allosteric inhibitor specificity of Thermotoga maritima 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway for the biosynthesis of aromatic amino acids. Allosteric regulation of Thermotoga maritima DAH7PS is mediated by L-Tyr binding to a discrete ACT regulatory domain appended to a core catalytic (ß/α)8 barrel. Variants of T. maritima DAH7PS (TmaDAH7PS) were created to probe the role of key residues in inhibitor selection. Substitution Ser31Gly severely reduced inhibition by L-Tyr. In contrast both L-Tyr and L-Phe inhibited the TmaHis29Ala variant, while the variant where Ser31 and His29 were interchanged (His29Ser/Ser31His), was inhibited to a greater extent by L-Phe than L-Tyr. These studies highlight the role and importance of His29 and Ser31 for determining both inhibitory ligand selectivity and the potency of allosteric response by TmaDAH7PS.

Neisseria meningitidis expresses a single 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase that is inhibited primarily by phenylalanine.

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.

Engineering allosteric control to an unregulated enzyme by transfer of a regulatory domain.

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-D-arabino-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.

Dynamic cross-talk among remote binding sites: the molecular basis for unusual synergistic allostery.

Allosteric regulation of protein function is critical for metabolic control. Binding of allosteric effectors elicits a functional change in a remote ligand binding site on a protein by altering the equilibrium between different forms in the protein ensemble. 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step in the shikimate pathway, which is responsible for the biosynthesis of aromatic amino acids Trp, Phe, and Tyr. Feedback regulation by the aromatic amino acids is important for controlling the cellular levels of the aromatic amino acids, and many organisms have two or more DAH7PS isozymes that show differing sensitivities to aromatic compounds. Mycobacterium tuberculosis expresses a single DAH7PS that is insensitive to the presence of a single amino acid yet shows extraordinary synergistic inhibition by combinations of the pathway end products Trp and Phe. The Trp+Phe-bound structure for M. tuberculosis DAH7PS, showing two separate binding sites occupied by Trp and Phe for each monomer of the tetrameric protein, was obtained by cocrystallization. Comparison of this structure with the ligand-free M. tuberculosis DAH7PS demonstrates that there is no significant change in conformation upon ligand binding, suggesting that contributions from altered dynamic properties of the enzyme may account for the allosteric inhibition. Isothermal titration calorimetry experiments demonstrate that the inhibitor binding sites are in direct communication. Molecular dynamics simulations reveal different changes in dynamic fluctuations upon single ligand binding compared to dual ligand binding. These changes account for the cross-talk between inhibitor binding sites and the active site, simultaneously potentiating both dual ligand binding and diminution of catalytic function.

Tyrosine latching of a regulatory gate affords allosteric control of aromatic amino acid biosynthesis.

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 (ß/α)(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.