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El piridoxal 5'-fosfato es la forma biocatalíticamente activa de la vitamina B6, siendo uno de los cofactores más versátiles de la naturaleza, el cuál tiene un papel central en el metabolismo de aminoácidos. Mientras que la mayoria de microorganismos y plantas pueden sintetizar la vitamina B6 de novo, los mamíferos se ven obligados a obtener uno de sus vitámeros a través de la dieta. La maquinaria biosintética de Escherichia coli es, de lejos, la mejor caracterizada y consiste en cuatro proteínas pdx. PdxJ, también conocida como piridoxina 5'-fosfato sintasa, es la enzima clave en esta via. Cataliza el último paso, la complicada reacción de cierre del anillo entre 1-deoxi-D-xilulosa-5-fosfato y aminoacetona-3-fosfato para formar piridoxina 5'-fosfato. La comparación de secuencias de PdxJ entre espécies revela que existe un alto grado de conservación indicando así la enorme importancia fisiológica de esta enzima.Con el uso de un derivado de mercurio fue posible el resolver la estructura cristalina de la enzima de E. coli por el método del "single isomorphous replacement with anomalous scattering" y el refinar la estructura a 2.0 Å de resolución. El monómero corresponde al plegamiento TIM o barril (_/_)8, con la incorporación de tres hélices extra que median los contactos entre intersubunidades en el octámero. El octámero representa el estado fisiológicamente relevante, que fué observado tanto en el cristal como en solución, y que esta organizado como un tetrámero de dímeros activos. La caracterización de la estructura cristalográfica de la enzima con sustratos, análogos de sustrato y productos unidos permitió la identificación del centro activo y la propuesta de un mecanismo detallado. Los rasgos catalíticos más remarcables son: (1) el cierre del centro activo una vez se han unido los sustratos, de manera que el bolsillo de unión queda aislado del solvente y los intermediarios de la reacción quedan así estabilizados; (2) la existencia de dos sitios de unión de fosfato bien definidos; (3) y un canal de agua que penetra el núcleo del barril _ y permite liberar las moléculas de agua formadas durante la reacción.La cantidad de información presentada debería permitir el diseño de inhibidores de la piridoxina 5'-fosfato sintasa basados en su estructura. Es interesante el destacar que entre las bacterias que contienen el gen pdxJ se encuentran unos cuantos patógenos bien conocidos. La resistencia de bacterias contra antibióticos está aumentando cada vez más, hecho que se está convirtiendo en un auténtico problema. Por este motivo, es necesario el desarrollar medicamentos antibacterianos con un alto grado de especificidad y la piridoxina 5'-fosfato sintasa parece ser una diana muy prometedora. / Pyridoxal 5'-phosphate is the biocatalytically active form of vitamin B6, being one of nature's most versatile cofactors that plays a central role in the metabolism of amino acids. Whereas microorganisms and plants can synthetise vitamin B6 de novo, mammals have to obtain one of the B6 vitamers with their diet. The Escherichia coli biosynthetic machinery is the, by far, best characterised and it consists in four pdx proteins. PdxJ, also referred to as pyridoxine 5'-phosphate synthase, is the key enzyme in this pathway. It catalyses the last step, the complicated ring-closure reaction between 1-deoxy-D-xylulose-5-phosphate and aminoacetone-3-phosphate yielding pyridoxine 5'-phosphate. Sequence comparison of PdxJ from different species revealed a remarkable high degree of conservation indicating the paramount physiological importance of this enzyme.With the use of one mercury heavy-atom derivative, it was possible to solve the crystal structure of the E. coli enzyme by the single isomorphous replacement method with anomalous scattering and to refine the structure at 2.0 Å resolution. The monomer folds as a TIM or (_/_)8 barrel, with the incorporation of three extra helices that mediate intersubunits contacts within the octamer. The octamer represents the physiological relevant state that was observed in the crystal and in solution, and that is organised as a tetramer of active dimers. Characterisation of the enzyme crystal structure with bound substrates, substrate analogues, and products allowed the identification of the active site and the proposal of a detailed reaction mechanism. The most important catalytic features are: (1) active site closure upon substrate binding, in order to isolate the specificity pocket from the solvent und thus stabilise the reaction intermediates; (2) the existence of two well-defined phosphate binding sites; (3) and a water channel that penetrates the _ barrel core and allows the release of waters in the closed state.The amount of information here presented should permit the structure-based design of pyridoxine 5'-phosphate synthase inhibitors. Interestingly, among bacteria that contain the pdxJ gene there are several well-known pathogens. More and more, the bacterial resistance against antibiotics is increasing and therefore becoming a real problem. Thus, it is necessary the development of highly specific antibacterial drugs and pyridoxine 5'-phosphate synthase seems to be a promising novel target.

Pyridoxal 5'-phosphate (PLP) is the cofactor for over 140 vitamin B6 (PLP)-dependent enzymes that are involved in various metabolic and biosynthetic pathways. Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two key enzymes that metabolize nutritional forms of vitamin B6, including pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) to the active cofactor form, PLP. Disruption of the PLP metabolic pathway due to mutations in PNP oxidase or PL kinase result in PLP deficiency, which is implicated in several neurological pathologies. Several ingested compounds are also known to result in PLP deficiency with concomitant neurotoxic effects. How these mutations and compounds affect B6 metabolism is not clearly understood. On the other hand, an emerging health problem is the intake of too much vitamin B6 as high doses of the reactive PLP in the cell exhibits toxic effects, including sensory and motor neuropathies. The overall aim of this research is to understand the catalytic function of PL kinase and the regulatory pathway of PLP metabolism. Using site-directed mutagenesis (Asp235Asn, Asp235Ala), kinetic and structural studies, we have shown that Asp235 may play a catalytic role in PL kinase phosphorylation activity. We also show that human PL kinase binds its substrates, PL and MgATP synergistically, and that the enzyme requires Na+ (or K+) and Mg2+ for its activity. Using kinetic study, we show severe induced MgATP substrate inhibition of PL kinase in the presence of its product, PLP, and we postulate this to be due to the formation of a non-productive ternary complex (Enzyme•PLP•MgATP). Consistently, our crystal structure of human PL kinase (2.1 Å) co-crystallized with MgATP and PLP showed both MgATP and PLP trapped at the active site. Our hypothesis is that this abortive ternary complex might be a physiological process, and that PL kinase uses this mechanism to self-regulate its activity. Our inhibition studies show theophylline, a bronchodilator as a mixed competitive inhibitor of human PL kinase with Ki of 71 μM. Our structural study (2.1 Å) shows theophylline bound at the substrate, PL binding site of human PL kinase. We also identified several potential PL kinase inhibitors from the DrugBank Chemical Compound database. Some of these compounds, including enprofylline, theobromine, caffeine, and lamotrigine, which incidentally exhibit similar neurotoxic effects as theophylline, show significant inhibitory effect on human PL kinase. Further studies are also planned to investigate the effect of these drugs on vitamin B6 metabolism in vivo.

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are two of four homodimeric members of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. The evolutionary relationships among α-oxoamine synthases representing a broad taxonomic and phylogenetic spectrum have been examined to help identify residues that may regulate substrate specificity. The structural plasticity of ALAS has been documented in studies of functional circularly permuted ALAS variants and the single polypeptide chain ALAS dimer (ALAS/ALAS) exhibiting a greater turnover number than wild-type ALAS. An examination of the contribution of each ALAS/ALAS active site to the enzymatic activity shows that each active site makes distinct contributions to the steady-state activity of the enzyme. Chimeric ALAS/AONS proteins exhibited an oligomeric structure with two sites having ALAS activity and two sites having AONS activity. Remarkably, the steady-state rates for both the ALAS and AONS activities were lower than that observed in the parent enzymes, while the reactivity of the ALAS sites in ALAS/AONS was similar to that of wild-type ALAS. We propose that the different contribution of each active site to the steady-state activity of ALAS/ALAS and the reduced steady-state activities of the ALAS/AONS chimera, compared to the parent enzymes, relate to different extents of conformational changes associated with product release due to the strain caused with the linking the two ALAS (or ALAS and AONS) subunits. Thus, the extensive plasticity seen in ALAS extends to another member of the α-oxoamine family, AONS. In the α-oxoamine synthase family a conserved histidine hydrogen bonds with the phenolic oxygen of PLP and may be significant for substrate-binding, PLP-positioning, and maintaining the pKa of the imine nitrogen. The replacement of this conserved histidine, H282, with alanine in murine erythroid ALAS has multiple effects on the spectral, binding, and kinetic properties of the enzyme and supports the conclusion that H282 plays multiple roles in the enzymology of ALAS. Altogether, these results imply that amino acid H282 coordinates the movement of the pyridine ring with the reorganization of the active-site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.

Amino acid biosynthesis is an essential process in living organisms. Certain amino acids can be synthesized by some organisms but not by others. L-Lysine is one of the essential amino acids that bacteria can synthesize but humans cannot. This is somewhat inconvenient for humans as much of their L-lysine must come from their diet. However, the lack of the lysine biosynthetic pathway in humans makes the bacterial enzymes within the pathway attractive drug targets. Recently, a novel lysine biosynthetic pathway was discovered in plants, Chlamydiae and some archaea. It is called the diaminopimelate aminotransferase (DAP-AT) pathway. In this pathway, LL-DAP-AT plays a key role by directly converting L-tetrahydrodipicolinate to LL-DAP in a single step. This is a quite interesting characteristic of LL-DAP-AT as the above conversion takes three sequential enzymatic steps in the previously known lysine biosynthetic pathways. Due to its absence in humans, LL-DAP-AT would be an attractive target for the development of novel antibiotics. In order to understand the catalytic mechanism and substrate recognition of LL-DAP-AT, the structural characterization of LL-DAP-AT is of paramount importance. In this thesis, the overall architecture of LL-DAP-AT and its substrate recognition mechanism revealed by the crystal structures of LL-DAP-AT from Arabidopsis thaliana and Chlamydia trachomatis will be discussed. The crystal structure of the native LL-DAP-AT from A. thaliana (AtDAP-AT) presented in this thesis is the first structure of LL-DAP-AT to be determined. This structure revealed that LL-DAP-AT forms a functional homodimer and belongs to the type I fold family of PLP dependent aminotransferases. The subsequent determination of the substrate-bound AtDAP-AT structure showed how the two substrates, (LL-DAP and L-Glu) significantly different in size, are recognized by the same set of residues without significant conformational changes in the backbone structure. In addition, the LL-DAP-bound AtDAP-AT structure shows that the C-amino group of LL-DAP is recognized stereospecifically by the active site residues that are unique to the family of LL-DAP-AT enzymes. Lastly, the chlamydial LL-DAP-AT presented in this thesis shows a new open conformation for LL-DAP-AT. The implications of the conformational flexibility of CtDAP-AT on the differences in substrate specificities among LL-DAP-AT are discussed.

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5-Aminolevulinate synthase (ALAS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation between glycine and succinyl-CoA to generate coenzyme A (CoA), CO2, and 5-aminolevulinate (ALA). The chemical mechanism of this reaction, which represents the first and regulated step of heme biosynthesis in mammals, involves the formation of a short-lived glycine quinonoid intermediate and an unstable 2-amino-3-ketoadipate intermediate. Using liquid chromatography coupled with tandem mass spectrometry to analyze the products from the reaction of murine erythroid ALAS (mALAS2) with O-methylglycine and succinyl-CoA, we directly identified the chemical nature of the inherently unstable 2-amino-3-ketoadipate intermediate, which predicates the glycine quinonoid species as its precursor. With stopped-flow absorption spectroscopy, we detected and confirmed the formation of the quinonoid intermediate upon reacting glycine with ALAS. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, L-serine was reacted with ALAS, a lag phase was observed in the progress curve for the L-serine external aldimine formation, indicating a hysteretic behavior in ALAS. Hysteresis was not detected in the T148A-catalyzed L-serine external aldimine formation. These results with T148A, a mALAS2 variant, which, in contrast to the wild-type enzyme, is active with L-serine, suggest that the active site T148 modulates the strict amino acid substrate specificity of ALAS. The rate of ALA release is also controlled by a hysteretic kinetic mechanism (observed as a lag in the ALA external aldimine formation progress curve), consistent with conformational changes governing the dissociation of ALA from ALAS. In Rhodobacter capsulatus ALAS, apart from coordinating the positioning of succinyl-CoA, N85 has an important role in regulating the opening of an active site channel. Here, we have mutated the analogous asparagine of murine erythroid ALAS to a histidine (N150H) and assessed its effects on catalysis through steady-state and pre-steady-state kinetic studies. Quinonoid intermediate formation occurred with a significantly reduced rate for the N150H-catalyzed condensation of glycine with succinyl-CoA during a single turnover. When the same forward reaction was examined under multiple turnovers, the progress curve of the N150H reaction displayed a prolonged decay of the quinonoid intermediate into the steady-state, distinct from the steep decay in the wild-type ALAS reaction. This prolonged decay results from an accelerated transformation of the product, ALA, into the quinonoid intermediate during the reverse N150H-catalyzed reaction. In fact, while wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold lower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the rate for the N150H-catalyzed reverse reaction is 1.7-fold higher than that of the forward reaction. We conclude that N150 is important in establishing a catalytic balance between the forward and reverse reactions, by favoring ALA synthesis over its non-productive transformation into the quinonoid intermediate. Mutations at this position could perturb the delicate heme biosynthetic equilibrium. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0-3.0 and 7.5-10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphtalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the kcat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.

Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Biochemistry / Unrestricted

In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be  an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially contributing to the biosynthesis of taurine and/or beta-alanine in mammalian species. Finally, the metabolic engineering work of L-3, 4-dihydroxyphenylalanine decarboxylase (DDC) and 3, 4-dihydroxylphenylacetaldehyde (DHPAA) synthase has revealed that the reactions of these enzymes could be determined by a few conserved residues at their active site. As both enzymes have been implicated in the biosynthesis of sclerotizing reagents, it is of great scientific and practical importance to understand the similarity and difference in their reaction mechanisms. The results of this dissertation provide valuable biochemical information of ADC, DDC, DHPAA synthase, and GADL1, all of which are PLP-dependent decarboxylases. ADC, DDC, DHPAA synthase are important enzymes in insect cuticle hardening by contributing to the biosynthesis of sclerotizing reagents. Knowledge towards understanding of these enzymes will promote the comprehension of insect cuticle hardening and help scientists to search for ideal insecticide targets. The characterization of GADL1 lays groundwork for future research of its potential role in taurine and beta-alanine metabolism. / Ph. D.

The biosynthesis of tetrapyrolles in eukaryotes and the alpha-subclass of purple photosynthetic bacteria is controlled by the pyridoxal 5?-phosphate (PLP)-dependent enzyme, 5-aminolevulinate synthase (ALAS). Aminolevulinate, the universal building block of these macromolecules, is produced together with Coenzyme A (CoA) and carbon dioxide from the condensation of glycine and succinyl-CoA. The three-dimensional structures of Rhodobacter capsulatus ALAS reveal a conserved active site serine that moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed, substrate-bound enzyme conformation, and simultaneously to within 3-4 angstroms of the thioester sulfur atom of bound succinyl-CoA. To elucidate the role(s) this residue play(s) in enzyme activity, the equivalent serine in murineerythroid ALAS was mutated to threonine or alanine. The S254A variant was active, but both the KmSCoA and kcat values were increased, by 25- and 2-fold, respectively, suggesting the increase in turnover is independent of succinyl-CoA-binding. In contrast, substitution of S254 with threonine results in a decreased kcat, however the Km for succinyl-CoA is unaltered. Removal of the side chain hydroxyl group in the S254A variant notably changes the spectroscopic properties of the PLP cofactor and the architecture of the PLP-binding site as inferred from circular dichroism spectra. Experiments examining the rates associated with intrinsic protein fluorescence quenching of the variant enzymes in response to ALA binding show that S254 affects product dissociation. Together, the data led us to suggest that succinyl-CoA binding in concert with the hydrogen bonding state of S254 governs enzyme conformational equilibria. As a member of the alpha-oxoamine synthase family, ALAS shares a high degree of structural similarity and reaction chemistry with the other enzymes in the group. Crystallographic studies of the R. capsulatus ALAS structure show that the alkanoate component of succinyl-CoA is bound by a conserved arginine and a threonine. To examine acyl-CoA-binding and substrate discrimination in murine erythroid ALAS, the corresponding residues (R85 and T430) were mutated and a series of CoA substrate analogs were tested. The catalytic efficiency of the R85L variant with octanoyl-CoA was 66-fold higher than that calculated for the wild-type enzyme, suggesting this residue is strategic in substrate binding. Hydrophobic substitutions of the residues that coordinate acyl-CoA-binding produce ligand-induced changes in the CD spectra, indicating that these amino acids affect substrate-mediated changes to the microenvironment of the chromophore. Pre-steady-state kinetic analyses of the R85K variant-catalyzed reaction show that both the rates associated with product-binding and the parameters that define quinonoid intermediate lifetime are dependent on the chemical composition of the acyl-CoA tail. Each of the results in this study emphasizes the importance of the relationship between the bifurcate interaction of the alkanoic acid component of succinyl-CoA and the side chains of R85 and T430. From the X-ray crystal structures of Escherichia coli 8-amino-7-oxonoanoate synthase and R. capsulatus ALAS, it was inferred that a loop covering the active site moved 3-6 Å between the holoenzymic and acyl-CoA-bound conformations. To elucidate the role that the active site lid plays in enzyme function, we shuffled the portion of the murine erythroid ALAS cDNA corresponding to the lid sequence (Y422-R439), and isolated functional variants based on genetic complementation in an ALA-deficient strain. Variants with potentially greater enzymatic activity than the wild-type enzyme were screened for increased porphyrin overproduction. Turnover number and the catalytic efficiency of selected functional variants with both substrates were increased for each of the enzyme variants tested, suggesting that increased activity is linked to alterations of the loop motif. The results of transient kinetics experiments for three isolated variants when compared to wild-type ALAS showed notable differences in the pre-steady-state rates that define the kinetic mechanism, indicating that the rate of ALA release is not rate-limiting for these enzymes. The thermodynamic parameters for a selected variant-catalyzed reaction indicated a reduction in the amount of energy required for catalysis. This finding is consistent with the proposal that, in contrast to the wild-type ALAS reaction, a protein conformational change associated with ALA release no longer limits turnover for this variant enzyme.