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 reaction of di­chloro­dimethyl­silane with a polydentate Schiff base ligand derived from pyridoxal and 2-ethano­lamine yielded the macrocyclic silicon compound (8E,22E)-4,4,12,18,18,26-hexa­methyl-3,5,17,19-tetra­oxa-8,13,22,27-tetra­aza-4,18-disilatri­cyclo­[22.4.0.010,15]octa­cosa-1(24),8,10,12,14,22,25,27-octa­ene-11,25-diol, C24H36N4O6Si2. The asymmetric unit contains the half macrocycle with an intra­molecular O—H⋯N hydrogen bond between the imine nitro­gen atom and a neighbouring oxygen atom. The crystal structure is dominated by C—H⋯O and C—H⋯π inter­actions, which form a high ordered mol­ecular network.

The work in this thesis is divided into two aims. The first aim is to provide a detailed analysis of water molecules at protein-protein interfaces as well as quantifying their contributions with respect to different residue types. To achieve this aim a data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å) X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on HINT. The second aim is to observe the effect of adding interfacial water molecules in developing a model for the protein-protein interaction between pyridoxal kinase and serine hydroxymethyltransferase. This model was created to explore the possibility of the formation of a channel between the two proteins upon interaction providing a safe way to transport the substrate pyridoxal 5’-phosphate (active form of vitamin B6). This work demonstrates a substantial progress in the understanding of the role of water molecules in protein-protein binding.

Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...

A malaria humana é uma doença infecciosa transmitida por um mosquito que está atrelada a uma alta taxa de mortalidade. Dentre os parasitas que causam a malaria humana a espécie Plasmodium falciparum é responsável por 90% dos casos letais. Sua virulência é ocasionada por modificações na célula hospedeira provenientes do tráfego de proteínas para a superfície de eritrócitos infectados. Com o intuito de interferir com estas proteínas, aplicamos a técnica Cell- SELEX (do inglês: Systematic Evolution of Ligands by Exponential Enrichment). Após ciclos iterativos de seleção obtivemos moléculas de DNA simples- fita, conhecidas como aptâmeros, que possuem alta afinidade e especificidade contra proteínas alvo presentes na superfície de eritrócitos infectados por P. falciparum. Ensaios de pull- down usando aptâmeros selecionados em sistema de purificação por streptavidina-biotina seguidos por análises de espectrometria de massa revelaram a interação destas moléculas de DNA com proteínas como RIFIN, PfEMP, RAP1, entre outras incorporadas na superfície de eritrócitos infectados. Como consequência destas interações, os aptâmeros selecionados demostraram ser inibidores potenciais na proliferação dos parasitas, como demonstrado por testes in vitro. Outra estratégia usada foi a produção de aptâmeros contra a piridoxal quinase de P. falciparum (PfPdxK) expressa. Usando tanto SELEX-proteína, bem como, ensaios de eletroforese capilar, selecionamos aptâmeros com capacidade para inibir a atividade enzimática da PfPdxK. Nossos dados constituem evidências sobre as aplicações da tecnologia SELEX no desenho racional de compostos contra a malária humana. / The human malaria is a mosquito-borne infectious disease associated with a high risk of mortality. Among the parasite species that causes human malaria, Plasmodium falciparum is responsible by 90% of the lethal cases. The virulence of this pathogen is associated with host cell modifications by trafficking proteins to the surface of infected erythrocytes. Aiming to interfere with these proteins we have applied the Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology. After iterative cycles of selection we obtained single stranded DNA molecules, known as aptamers, with high binding affinity and specificity against protein targets present in the surface of erythrocytes infected with P. falciparum. Pull-down assays using the selected aptamers in a streptavidin-biotin affinity assay followed by mass spectrometry analysis revealed the interaction of these DNA molecules with proteins such as RIFIN, PfEMP, RAP1, among others embedded in the surface of infected erythrocytes. As consequence of these interactions, the selected aptamers demonstrated to be potent inhibitors of parasite proliferation, as validated by in vitro tests. Another strategy used was the production of aptamers against the recombinantly expressed plasmodial pyridoxal kinase (PfPdxK). Using both protein-SELEX as well as capillary electrophoresis assays, we selected aptamers with capacity to inhibit the enzymatic activity of PfPdxK. Our data constitute evidences about the application of SELEX technology in the rationale design of inhibitors against human malaria.

O metabolismo de vitamina B9 é um alvo terapêutico conhecido para malária. A vitamina B6 foi validada como essencial para o parasita. Esse trabalho analisa bioquimica- e estruturalmente mutantes da enzima piridoxal quinase, pela combinação de análises in silico associadas a ensaios bioquímicos. A estrutura da PdxK de P. falciparum permitiu a localização do sítio ativo e da interface de dimerização. Logo foram sugeridas mutações que alterassem esses resíduos para investigar sua importância. Dinâmica molecular sugere que a dimerização é responsável pela estabilidade do sítio ativo, algo coerente com a diminuição da atividade enzimática na maioria das mutantes. Filtração em gel aponta um equilíbrio entre a conformação monômero-dímero mostrando que as mutações na interface não estão relacionadas a dimerização, mas envolvidas com a estabilização do folding na região do sítio ativo. A mesma é recoberta por uma tampa que impede a auto-hidrólise do ATP, há também um resíduo serina que estabiliza a conformação do piridoxal durante a catálise, as mutações em ambas as regiões levaram a inativação da enzimática. A hipótese de que a interação da PfPdxK com ligantes de RNA não se mostrou conclusiva. / Vitamin B9 metabolism is a known drug-target for malaria. Vitamin B6 was validated as essential for the parasite. We analysed biochemically and structurally the plasmodial dimeric pyridoxal kinase. PfPdxKs allowed us to determine the localisation of the active site as well the interface between the two monomers and to identify the involved residues. Molecular dynamics shows that the PdxKs dimerization is important for the active sites stability, which was confirmed by the decrease of activity in mutants related to this region. Gel filtration revealed equilibrium of monomer-dimer conformation and therefore the interface mutations decrease in activity might not be directly related to the dimerization processes, but rather to the active site organisation. The active site region shows a serine involved in keeping the pyridoxals conformation during catalyses and the identified lid region covering the ATP binding site is responsible for preventing auto-hydrolysis. Substitution of the respective amino acid to alanine resulted in enzyme inactivation. In silico analysis of the PfPdxK spacer region identified nucleic acid binding sides, however RNA binding experiments failed so far and the possibility of protein-RNA binding remains for elucidation.

Diaminopropionate ammonia lyase (DAPAL) which belongs to the  class of PLP enzymes is reported only from prokaryotes. It is involved in the removal of two amino groups from its substrate, diaminopropionate, to form ammonia and pyruvate. DAPAL from Escherichia coli (eDAPAL) and Salmonella typhimurium (sDAPAL) was cloned, over expressed and purified using either affinity chromatography or conventional procedures. It was observed that eDAPAL (90 units / mg) was comparatively less active than sDAPAL (200 units / mg). Also the enzymes with the N-terminal His tag were found to be many fold less active than the enzymes without tag. DAPAL had a characteristic absorption maximum at 414nm due to the Schiff`s linkage between PLP and the € - amino group of the active site lysine residue. The apoenzyme was prepared by reaction with L-cysteine, and the resulting thiazolidine complex was easily dialyzed. On reconstitution with PLP, complete regain of absorption spectrum and 60% activity was seen. All the three enzymes (apo-, holo and reconstituted), when subjected to gel filtration chromatography were found to be homodimers of 88 kDa. The active site lysine 78 was mutated to glutamine, and the enzyme was purified to homogeneity. In the mutant enzyme PLP continued to be bound at the active site, but in a different orientation with an absorbance maximum at 406nm. The K78Q enzyme had negligible activity as compared to the wild type enzyme confirming the role of K78 in catalysis. Only a few of the enzymes of the  class have been investigated for their unfolding pathways. Urea induced unfolding studies on sDAPAL revealed that at lower concentrations of urea there was a loss in activity due to the disruption of Schiff's linkage. No gross conformational changes were observed at these concentrations of urea as seen from fluorescence and gel filtration experiments. Increase in concentration of urea led to unfolding of the protein thereby causing a shift in fluorescence maximum from 340nm to 357 nm due to the exposure of the buried tryptophans to the less hydrophobic environment. A considerable amount of aggregation was seen at intermediate urea concentrations, which was possibly the reason for the inability of the protein to refold completely. Based on the results, a concerted mechanism for dissociation and unfolding was proposed for sDAPAL. Aminooxy compounds, which are mechanism-based inhibitors for PLP enzymes have been used as drugs against various disorders for the last few decades. In order to probe the mechanism and efficiency with which these compounds inhibit sDAPAL, cycloserine (D and L), methoxyamine (MA) and aminooxyacetic acid (AAA) were chosen for the inhibition studies. The inhibition rates were measured by monitoring decrease in absorbance at 414nm, increase in the range of 320-330nm due to the product formation and loss of activity upon incubation with the inhibitor. It was seen that both the enantiomers of cycloserine were equally effective in disrupting the Schiff’s linkage with the second order rate constants of 15.8 and 36 M -1 sec –1 respectively. Spectral measurements showed two isosbestic points in the case of DCS and one in the case of LCS. Product of this inhibition reaction was identified to be a heat and acid stable compound namely a hydroxyisooxazole derivative of PMP. It was similar in nature to that reported from GABA aminotransferase. These results showed that unlike in the case of alanine racemase, sDAPAL could be inhibited equally well by both the enantiomers. The inhibition studies with the other two inhibitors namely AAA and MA, showed AAA to be more efficient at disrupting the Schiff’s linkage and causing inactivation of the enzyme. The visible absorbance spectrum showed a single isosbestic point in both the cases, indicative of a single step involved in the formation of the final product. The elution profile of the product of the enzymatic as well as non-enzymatic reactions on a C-18 HPLC column was similar and the product was identified to be an oxime. These inhibitors reacted with sDAPAL many fold better than the other PLP dependent enzymes and therefore these compounds can serve as potential drugs for sDAPAL.

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.