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Vitamin B6 metabolism and regulation of pyridoxal kinaseGandhi, Amit K., January 1900 (has links)
Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Medicinal Chemistry. Title from title-page of electronic thesis. Bibliography: leaves 77-92.
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Pyridoxal Phosphate as a Tag to Identify Enzymes Within the “PLP-ome”Messer, Kayla J. 2011 May 1900 (has links)
The main objective of this research was to develop a protocol in which pyridoxal phosphate (PLP) would act as a tag to identify PLP-dependent enzymes from complex mixtures or cell lysates. Following the purification of a PLP-dependent enzyme (CysM), a method was developed to reduce the PLP-lysine Schiff base to form a chemically stable bond between the PLP and the protein. The reduced protein was enzymatically digested resulting in multiple peptide fragments with one or more containing PLP (bound to the active site lysine). These fragments were analyzed by monitoring the absorbance or fluorescence using High Performance Liquid Chromatography. Immobilized Metal Ion Affinity Chromatography (IMAC) was then used to enrich the PLP-peptide(s) from the peptide mixture. The PLP-bound peptide(s) was then analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS).
More specifically, sodium borohydride (NaBH4) was used to reduce the Lysine-PLP bond in CysM. This reaction was monitored by either UV-vis spectroscopy or mass spectrometry. Trypsin was used to enzymatically digest the reduced CysM before it was enriched with IMAC and analyzed with LC-MS. Since the objective of this project was to develop a method which could be applied to a cell lysate, IMAC was used as an enrichment method to separate the PLP-peptide(s) from other peptides within the mixture. The PLP-peptide(s) was then located in the peptide mixture by monitoring the absorbance at 325 nm. The LC-MS results of the full reaction before IMAC treatment versus the final column, when monitoring the mass spectrum, showed that the treatment using the IMAC column separated the PLP-peptides from all other peptides within the sample. Using IMAC to enrich specifically the PLP-peptides, followed by analysis with LC-MS, may be a useful method for studying PLP-dependent enzymes within the proteome or the "PLP-ome."
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The Effects of Vitamin B6 Supplementation on Mood States in College Women Taking Oral ContraceptivesJanuary 2020 (has links)
abstract: Oral contraceptives are one of the most frequently used forms of birth control among young women. However, research has shown that this type of medication can contribute to negative changes in mood and diminished vitamin status. In particular, women taking oral contraceptives are at an increased risk of vitamin B6 deficiency due to changes in enzyme activity with estrogen intake. Depressed mood is one of the known symptoms of vitamin B6 deficiency as this vitamin acts as an essential cofactor in converting tryptophan to the neurotransmitter, serotonin. Lack of adequate levels of vitamin B6 therefore contribute to decreased production of serotonin and subsequent changes in mood, including symptoms of depression. With vitamin B6 being the most common nutrient deficiency, and the ever increasing prevalence of depression in the United States, especially among young adults, it is crucial that researchers investigate ways to mitigate both of these undesirable side effects. Current research on the topic fails to directly connect supplementation of vitamin B6 to positive changes in mood in oral contraceptive users.
This 12-week long double-blinded, placebo-controlled crossover trial examined the effects of daily supplementation of vitamin B6 as 100 mg of pyridoxine hydrochloride, on mood states in 8 healthy college women (18-25 y) that use combined oral contraceptives. Vitamin status was assessed via plasma pyridoxal 5’-phosphate (PLP). Plasma PLP levels significantly increased by >193% (p=0.003) with daily supplementation of 100 mg B6 over a four week period. Mood changes with supplementation were assessed using the Profile of Mood States (POMS). Although a small improvement in the POMS depression sub score was observed after 4 weeks of vitamin B6 supplementation (14.7%), the changes were insignificant (p>0.05). Furthermore, total mood disturbance scores did not significantly change with either the placebo or supplement periods. While mood states were not improved, a significant decrease in the presence of depressive symptoms as measured by the Beck Depression Inventory was observed after vitamin B6 supplementation, compared to placebo (p=0.047). The results of this study necessitate further investigation into the use of B6 supplementation as a means of reducing negative mood changes in oral contraceptive users. / Dissertation/Thesis / Masters Thesis Nutrition 2020
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Structural and mechanistic studies of the pyridoxal 5'-phosphate-dependent enzyme serine palmitoyltransferaseMykhaylyk, Bohdan January 2018 (has links)
Sphingolipids (SLs) are complex lipid-derived structures that are essential components of cell membranes in eukaryotes and some bacteria. SLs and their complex derivatives ceramides are known to be involved in multiple processes such as the formation of lipid rafts, cell signalling and membrane trafficking. The first step of SL biosynthesis is universal to all sphingolipid-producing organisms from bacteria to humans and is catalysed by the enzyme serine palmitoyltransferase (SPT). SPT is a member of the alpha-oxoamine synthase (AOS) family of pyridoxal- 5'-phosphate-dependent enzymes. All AOS family enzymes retain a high degree of structural homology and catalyse the decarboxylative Claisen-like condensation of amino acids with thioester substrates. The SPT enzyme catalyses the formation of the universal SL precursor, 3-ketodihydrosphingosine (KDS), by condensation of L-serine and coenzyme A-derived palmitic acid. Being the key controller in SL biosynthesis, SPT plays a big role in regulating natural and pathological processes. A lot of research interest has been recently generated by SLs isolated from bacterial members of the human microbiome and their roles in human health. Increasing evidence suggests that some of these SLs possess immunoregulatory effects and can have a direct impact on the immunity of the host. Bacteroides fragilis is a commensal gut-dwelling bacterium that belongs to a few human microbionts known to produce unique iso-branched sphingolipids (isoSLs); these have been shown to influence the human iNKT cell count. The production of SLs in B.fragilis is completely regulated by a gene product BF2461. In this work, BF2461 was expressed and purified; using a combination of UV-vis spectrometry, enzymatic assays, mass spectrometry and protein X-ray crystallography, it has been confirmed to be an SPT. The substrate specificity of the BfSPT has been assessed with a range of different chain-length substrates, including less common 15 and 17-carbon chain length coenzyme A substrates. The enzyme can produce different types of SL precursors with a preference for the 16-carbon chain substrate palmitoyl- CoA. However, at high levels of PCoA, a substrate inhibition is observed that might point to a natural control mechanism employed by the bacterium in favour of producing iso-branched SLs (isoSLs). The structure of BfSPT has been elucidated in a complex with its amino acid substrate L-serine. Search and analysis of putative SPTs from other microbiome-associated bacteria that produce isoSLs show that they share high similarity with an average amino acid conservation of 74%, suggesting they might be adapted to a particular type of substrate. In this respect, BfSPT might be the first isoSL-producing SPT to be structurally characterised, and the first one to have a direct impact on human health. Further structural data were obtained on protein complexes with L-cycloserine and L-penicillamine, some common inhibitors of the PLP-dependent enzymes. The structure obtained in the presence of L-penicillamine provides the first direct structural evidence of the inhibitory mechanism by a thiazolidine complex formation in the active site of a PLP-dependent enzyme. These findings shed light on certain aspects of the reaction and inhibition mechanisms of BfSPT as well as opening new prospects into researching this interesting target and its impact on the human microbiome.
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Structural and Functional Studies on Pyridoxal 5′-Phosphate Dependent Lyases and AminotransferasesBisht, Shveta January 2013 (has links) (PDF)
The thesis describes structural and functional studies of two PLP-dependent enzymes, diaminopropionate (DAP) ammonia lyase (DAPAL) and N-acetylornithine aminotransferase (AcOAT). The main objective of this work was to understand the structural features that control and impart specificity for PLP-dependent catalysis.
DAPAL is a prokaryotic enzyme that catalyzes the degradation of D and L forms of DAP to pyruvate and ammonia. The first crystal structure of DAPAL was determined from Escherichia coli (EcDAPAL) in holo and apo forms, and in complex with various ligands. The structure with a transient reaction intermediate (aminoacrylate-PLP azomethine) bound at the active site was obtained from crystals soaked with substrate, DL-DAP. Apo and holo structures revealed that the region around the active site undergoes transition from disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. Based on the crystal structures and biochemical studies, as well as studies on active site mutant enzymes, a two base mechanism of catalysis involving Asp120 and Lys77 is suggested.
AcOAT is an enzyme of arginine biosynthesis pathway that catalyses the reversible conversion of N-acetylglutamate semialdehyde and glutamate to N-acetyl ornithine and α-ketoglutarate. It belongs to subgroup III of fold type I PLP dependent enzymes. Many clinically important aminotransferases belong to the same subgroup and share many structural similarities. We have carried out extensive comparative analysis of these enzymes to identify the unique features important for substrate specificity. Crystal structures of AcOAT from Salmonella typhimurium were determined in presence of two ligands, canaline and gabaculine, which are known to act as general inhibitors for most of the enzymes of this class. There structures provided important insights into the mode of binding of the substrates. The structures illustrated the switching of conformation of an active site glutamate side chain on binding of the two substrates. In addition to that, structural transitions involving three loop regions near the active site were observed in different ligand bound structures. Kinetics of single turnover fast reactions and multiple turnover steady state reactions indicated that N-AcOAT dimer might follow a mechanism involving sequential half site reactivity for efficient catalysis. The changes observed in loop conformation that resulted in asymmetric forms of the dimer enzyme might form the structural basis for half site reactivity. Single site mutants were designed to understand the significance of these structural transitions and the specific role of active site residues in determining substrate specificity and catalysis. Biochemical characterization of wild type and mutant enzymes by steady state and fast kinetic studies, along with their crystal structures provided detailed insights into subtlety of active site features that manifest substrate specificity and catalytic activity.
The thesis also describes the investigations on fold type II enzymes directed towards analyses of polypeptide folds of these enzymes, features of their active sites, nature of interactions between the cofactor and the polypeptide, oligomeric structure, catalytic activities with various ligands, origin of specificity and plausible regulation of activity. Analysis of the available crystal structures of fold type II enzymes revealed five different classes. The dimeric interfaces found in these enzymes vary across the classes and probably have functional significance.
Contributions made towards structural and functional studies of three other PLP-dependent enzymes, serine hydoxymethyltransferase (SHMT), D-serine deaminase (DSD) and D-cysteine desulfhydrase (DCyD) are described in an appendix.
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Inhibitory myší serinracemasy / Inhibitors of mouse serine racemaseVorlová, Barbora January 2013 (has links)
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...
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Diaminopropionate Ammonia Lyase : Characterization, Unfolding And Mechanism Of Inhibition By Aminooxy CompoundsKhan, Farida 03 1900 (has links)
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.
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Structural Studies On Pyridoxal 5'-Phosphate Dependent Enzymes Involved In D-Amino Acid Metabolism And Acid Tolerance ReponseBharath, S R 06 1900 (has links) (PDF)
Metabolism of D-amino acids is of considerable interest due to their key importance in cellular functions. The enzymes D-serine dehydratase (DSD) and D-cysteine desulfhydrase (DCyD) are involved in the degradation of D-Ser and D-Cys, respectively. We determined the crystal structure of Salmonella typhimurium DSD (StDSD) by multiple anomalous dispersion method of phasing using selenomethione incorporated protein crystals. The structure revealed a fold typical of fold type II PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild type StDSD (WtDSD) and selenomethionine labeled StDSD (SeMetDSD), significant electron density was not observed for the co-factor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other fold type II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modeling suggested that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β-elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates.
Salmonella typhimurium DCyD (StDCyD) is a fold type II PLP-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. We determined the crystal structure of StDCyD using molecular replacement method in two different crystal forms. The better diffracting crystal form obtained in presence of benzamidine illustrated the influence a small molecule in altering protein interfaces and crystal packing. The polypeptide fold of StDCyD consists of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) which resemble other fold type II PLP-dependent enzymes. X-ray crystal structures of StDCyD were also obtained in the presence of substrates, D-Cys and βCDA, and substrate analogs, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS). The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggested formation of PMP by the hydrolysis of cycloserines. Mutational studies suggested that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, we proposed a probable mechanism for the degradation of D-Cys by StDCyD.
The acid-induced arginine decarboxylase (ADC) is part of an enzymatic system in Salmonella typhimurium that contributes to making this organism acid resistant. ADC is a PLP-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced StADC to 3.1 Å resolution. StADC structure revealed an 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevent inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.
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Investigating the porphyrias through analysis of biochemical pathways.Ruegg, Evonne Teresa Nicole January 2014 (has links)
ABSTRACT
The porphyrias are a diverse group of metabolic disorders arising from diminished
activity of enzymes in the heme biosynthetic pathway. They can present with acute
neurovisceral symptoms, cutaneous symptoms, or both. The complexity of these
disorders is demonstrated by the fact that some acute porphyria patients with the
underlying genetic defect(s) are latent and asymptomatic while others present with
severe symptoms. This indicates that there is at least one other risk factor required in
addition to the genetic defect for symptom manifestation. A systematic review of the
heme biosynthetic pathway highlighted the involvement of a number of micronutrient
cofactors. An exhaustive review of the medical literature uncovered numerous reports
of micronutrient deficiencies in the porphyrias as well as successful case reports of
treatments with micronutrients. Many micronutrient deficiencies present with
symptoms similar to those in porphyria, in particular vitamin B6. It is hypothesized
that a vitamin B6 deficiency and related micronutrient deficiencies may play a major
role in the pathogenesis of the acute porphyrias. In order to further investigate the
porphyrias, a computational model of the heme biosynthetic pathway was developed
based on kinetic parameters derived from a careful analysis of the literature. This
model demonstrated aspects of normal heme biosynthesis and illustrated some of the
disordered biochemistry of acute intermittent porphyria (AIP). The testing of this
model highlighted the modifications necessary to develop a more comprehensive
model with the potential to investigated hypotheses of the disordered biochemistry of
the porphyrias as well as the discovery of new methods of treatment and symptom
control. It is concluded that vitamin B6 deficiency might be the risk factor necessary
in conjunction with the genetic defect to trigger porphyria symptoms.
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