The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.

The shikimate pathway is a primary metabolic pathway involved in the synthesis of aromatic compounds in plants, fungi, apicomplexan parasites and microbes. The absence of this pathway in animals makes it ideal for the synthesis of antimicrobial compounds and herbicides. Additionally, its branching into indole hormone synthesis and phenylpropanoid secondary metabolism makes this pathway attractive for metabolic engineering. Here, the focus is on the first step of the shikimate pathway catalyzed by DAHP synthase. This step consists of the condensation of phosphoenol pyruvate and erythrose-4-phosphate to make DAHP, which undergoes another six catalytic steps to synthesize chorismate, the precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP synthase isozymes, which are known to indirectly regulate downstream pathways in response to wounding and pathogen stress. The model presented here proposes that DAHP synthase isozymes are regulated by the end products tyrosine, tryptophan and phenylalanine.

Hypoxia encountered at high altitude (HA) can limit energy production via aerobic metabolism in animals. Carbohydrate oxidation (CHO) has a greater ATP yield/mole O2 than fat oxidation, and HA-native deer mice show an increased reliance on CHO during submaximal exercise after hypoxia acclimation as an O2-saving strategy. However, hypoxia acclimation does not increase glycolytic capacity in muscle. We therefore tested the hypothesis that altered metabolic regulation of the CHO pathway allows HA mice to achieve higher rates of CHO during submaximal exercise. The objective of our study was to identify the effects of hypoxia acclimation on the regulation of two key proteins in the CHO pathway and their activation with exercise. Using first generation (G1) laboratory born and raised HA deer mice acclimated to normoxia or chronic hypoxia, we examined the metabolic regulation of muscle glucose uptake by glucose transporter (GLUT) 4 and of pyruvate oxidation by pyruvate dehydrogenase (PDH). The gastrocnemius was electrically stimulated in situ under anaesthesia and acute normoxia at two submaximal workloads relative to maximal force production, which was measured using a force transducer. In frozen gastrocnemius following stimulation or rest, GLUT4 protein content was measured via Western blotting of the sarcolemmal membrane fraction and PDH activity was measured using a radiolabelled assay. We found no differences in sarcolemmal GLUT4 content with stimulation, but PDH activity was increased in hypoxia, indicating increased rates of carbohydrate breakdown at similar workloads after acclimation. These data were compared to data from wild HA deer mice sampled at their native altitude. In support of our hypothesis, these data show that the metabolic regulation of the carbohydrate oxidative pathway changes with acclimation to support higher CHO rates during submaximal exercise. These data will help uncover the mechanistic underpinnings responsible for the exercise fuel use strategies observed exclusively in HA-native mice. / Thesis / Master of Science (MSc) / At high altitude, oxygen availability is low and can be challenging for active animals. Preferential carbohydrate oxidation is a metabolic strategy used by high altitude-native deer mice to fuel exercise because of its high energy yield per oxygen consumed. Despite the increase in carbohydrate breakdown, the capacity for muscles to use carbohydrates did not change, suggesting that the regulation of this metabolic pathway may be changing instead. We measured the contributions of two proteins involved in carbohydrate metabolism in active muscle, pyruvate dehydrogenase (PDH) and glucose transporter 4 (GLUT4), at different muscle workloads and after acclimation to high altitude conditions. We found no differences in GLUT4 content, but PDH activity was higher in hypoxia-acclimated mice at similar intensities, indicating increased rates of carbohydrate breakdown after acclimation. These data suggest that the regulation of the carbohydrate metabolic pathway changes with acclimation to support higher rates of carbohydrate oxidation during exercise.

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Metabolic systems are open systems continually subject to changes in the surrounding environment that cause uctuations in the state variables and perturbations in the system parameters. However, metabolic systems have mechanisms to keep them dynamically and structurally stable in the face of these changes. In addition, metabolic systems also cope with large changes in the uxes through the pathways, not letting metabolite concentrations vary wildly. Quantitative measures have previously been proposed for "metabolic regulation", using the quantitative framework of Metabolic Control Analysis. However, the term "regulation" is so loosely used so that its content is mostly lost. These di erent measures of regulation have also not been applied to a model and comparably investigated prior to this study. Hence, this study analyses the usefulness of the di erent quantitative measures in answering di erent types of regulatory questions. Thus, the aim of this study was to distinguish the above mentioned aspects of metabolic regulation and to nd appropriate quantitative measures for each, namely dynamic stability, structurally stability, and homeostasis. Dynamic stability is the property of a steady state to return to its original state after a perturbation in a metabolite in the system, and can be analysed in terms of self and internal-response coe cients. Structural stability is concerned with the change in steady state after a perturbation of a parameter in the system, and can be analysed in terms of concentration-response coe cients. Furthermore, it is shown that control patterns are useful in understanding which system properties determine structural stability and to what degree. Homeostasis is de ned as the change in the steady-state concentration of a metabolite relative to the change in the steady-state ux through the metabolite pool following a perturbation in a system parameter, and co-response coe cients are proposed as quantitative measures of homeostasis. More speci cally, metabolite-ux coresponse coe cients allow the de nition of an index that quanti es to which degree a metabolite is homeostatically regulated. A computational model of a simple linear metabolic sequence subject to feedback inhibition with di erent sets of parameters provided a test-bed for the quantitative analysis of metabolic regulation. Log-log rate characteristics and parameter portraits of steady-state variables, as well as response and elasticity coe cients were used to analyse the steady-state behaviour and control properties of the system. This study demonstrates the usefulness of generic models based on proper enzyme kinetics to further our understanding of metabolic behaviour, control and regulation and has laid the groundwork for future studies of metabolic regulation of more complex core models or of models of real systems. / AFRIKAANSE OPSOMMING: Metaboliese sisteme is oop sisteme wat gedurig blootgestel word aan `n uktuerende omgewing. Hierdie uktuasies lei tot veranderinge in beide interne veranderlikes en parameters van metaboliese sisteme. Metaboliese sisteme besit egter meganismes om dinamies en struktureel stabiel te bly. Verder verseker hierdie meganismes ook dat die konsentrasies van interne metaboliete relatief konstant bly ten spyte van groot veranderinge in uksie deur die metaboliese pad waarvan hierdie metaboliete deel vorm. Kwantitatiewe maatstawwe is voorheen voorgestel vir "metaboliese regulering", gebaseer op die raamwerk van Metaboliese Kontrole Analise. Die onkritiese gebruik van die term "regulering" ontneem egter hierdie konsep van sinvolle betekenis. Voor hierdie studie is die voorgestelde maatstawwe van regulering nog nie toegepas op 'n model ten einde hulle met mekaar te vergelyk nie. Die huidige studie ondersoek die toepaslikheid van die verskillende maatstawwe om verskillende tipe vrae oor regulering te beantwoord. Die doelwit van hierdie studie was om aspekte van metaboliese regulering, naamlik dinamiese stabiliteit, strukturele stabiliteit en homeostase, te onderskei, asook om 'n gepaste maatstaf vir elk van die verskillende aspekte te vind. Dinamiese stabiliteit is 'n eienskap van 'n bestendige toestand om terug te keer na die oorspronklike toestand na perturbasie van die konsentrasie van 'n interne metaboliet. Hierdie aspek van regulering kan in terme van interne respons en self-respons koeffi siente geanaliseer word. Strukturele stabiliteit van 'n bestendige toestand beskryf die mate van verandering van die bestendige toestand nadat 'n parameter van die sisteem geperturbeer is, en kan in terme van konsentrasie-responskoeffisiente geanaliseer word. Verder wys hierdie studie dat kontrole patrone van nut is om vas te stel watter eienskappe van 'n sisteem die strukturele stabiliteit bepaal en tot watter mate. Homeostase word gede finieer as die verandering in die konsentrasie van 'n interne metaboliet relatief tot die verandering in die uksie deur daardie metaboliese poel nadat 'n parameter van die sisteem verander het. Vir die analise van hierdie aspek van regulering word ko-responskoe ffisiente as 'n maatstaf voorgestel. Meer spesi ek kan metaboliet- uksie ko-responskoeff siente gebruik word om `n indeks te de nieer wat meet tot watter mate 'n metaboliet homeostaties gereguleer word. 'n Rekenaarmatige model van 'n eenvoudige lineere metaboliese sekwens wat onderhewig is aan terugvoer inhibisie is gebruik om die verskillende aspekte van metaboliese regulering kwantitatief te analiseer met vier verskillende stelle parameters. Dubbel-logaritmiese snelheidskenmerke en parameter portrette van bestendige toestandsveranderlikes, asook van respons- en elastisiteit koeffisente is gebruik om die bestendige toestandsgedrag en kontrole eienskappe van die sisteem te analiseer. Hierdie studie demonstreer die nut van generiese modelle wat op korrekte ensiemkinetika gebaseer is om ons verstaan van metaboliese gedrag, kontrole en regulering te verdiep. Verder dien hierdie studie as grondslag vir toekomstige studies van metaboliese regulering van meer ingewikkelde kernmodelle of modelle van werklike sisteme. / National Research Foundation

One of the cellular responses to temperature fluctuations in plants is the adjustment in the degree of membrane unsaturation. Glycerolipids are major constituents of cellular membranes. In higher plants, glycerolipids are synthesized via two major metabolic pathways compartmentalized in the ER and chloroplast. Adaptive responses in membrane lipids include alterations in fatty acid desaturation, proportional changes in membrane lipids as well as molecular composition of each lipid species. In this study, I systematically explored the significance of glycerolipid pathway balance in temperature induced lipid composition changes in three plant species that have distinctive modes of lipid pathway interactions through a combination of biochemical and molecular approaches including lipidomics and RNA-seq analysis. In Arabidopsis thaliana, a 16:3 plant, low temperature induces an augmented prokaryotic pathway, whereas high temperature enhances the eukaryotic pathway. Atriplex lentiformis reduces its overall lipid desaturation at high temperature and switches lipid phenotype from 16:3 to 18:3 through drastically increasing the contribution of the eukaryotic pathway as well as suppression of the prokaryotic pathway. In sync with the metabolic changes, coordinated expression of glycerolipid pathway genes, as revealed by RNA-seq also occurs. In Triticum aestivum, an 18:3 plant, low temperature leads to a reduced glycerolipid flux from ER to chloroplast. Evidence of differential trafficking of diacylglycerol (DAG) moieties from ER to chloroplast was uncovered in three plant species as another layer of metabolic adaptation under different temperatures. Taken together, this study has established a biochemical basis that highlights the predominance and prevalence of lipid pathway interactions in temperature induced lipid compositional changes.

No description available.

Three 2-oxoglutarate-dependent dioxygenases required for flavonoid biosynthesis were characterized in Arabidopsis thaliana. Genes encoding flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), and leucoanthocyanidin dioxygenase (LDOX) were cloned and sequenced. The predicted proteins encoded by each of these Arabidopsis genes shared high homology with all F3H, FLS, or LDOX sequences available in Genbank. Low-stringency DNA blot analysis indicated that F3H and LDOX are encoded by a single gene in Arabidopsis, while FLS may be encoded by two or three genes. RNA blot analysis was performed to determine the expression patterns of these three genes relative to previously-cloned genes encoding flavonoid biosynthetic enzymes. Light-induction experiments and analysis of regulatory mutants showed that the CHS, CHI, F3H, and FLS1 are coordinately regulated in Arabidopsis seedlings, encode enzymes acting near the beginning of the pathway, and are therefore referred to as "early" genes. The same experiments showed that DFR and LDOX are regulated distinctly from "early" genes, share similar expression patterns in response to light, and are not expressed in the ttg mutant. DFR and LDOX are therefore referred to as "late" genes due to the timing of expression in response to light and the fact that they encode enzymes acting late in flavonoid biosynthesis. To determine whether any of the previously-identified transparent testa mutants were defective in F3H, FLS, or LDOX, the chromosomal locations of these genes in the Arabidopsis genome were determined. The positions of these genes suggested that no previously-identified tt mutant was defective in the cloned FLS or LDOX structural genes, while tt6 was potentially the F3H locus. The coding region of F3H was amplified by PCR from tt6 genomic DNA and sequenced, and several point mutations were found in the coding region of this allele, three of which are predicted to result in amino acid substitutions. Polyclonal antibodies were also developed using four different purified, recombinant flavonoid enzymes as antigens. These antibodies were used to determine the pattern of accumulation of flavonoid enzymes in developing seedlings. Immunoblot analysis was also performed to determine whether mutations in genes encoding specific flavonoid enzymes or an enzyme in pathways that compete for or provide substrate for flavonoid biosynthesis (mutants defective in tryptophan or ferulic acid biosynthesis) affect the levels of flavonoid enzymes. These analyses showed that mutant seedlings which lacked specific flavonoid or tryptophan biosynthetic enzymes accumulated higher steady-state levels of other enzymes in the pathway. These results suggest that the accumulation of specific flavonoid intermediates or indole can lead directly or indirectly to higher levels of flavonoid enzymes. / Ph. D.

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids. / Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon.

No description available.