Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Metabolic networks of cellular systems are complex, in that there are numerous components with multiple non-linear interactions. To understand how these networks work they are often split into manageable pieces and studied individually. However, an individual part is unable to account for the complex properties of systems. In order to study these interactions the eld of systems biology has developed. Systems biology makes use of computers to construct models as a method to describe aspects of living systems. Using cellular pathways, kinetic models of metabolic pathways can be constructed and used as a tool to study the biological systems and provide a quantitative description. This thesis describes the quantitative analysis of a bacterium using a systems biology approach. Zymomonas mobilis is a rod shaped, Gram-negative, non-mobile facultative anaerobe and has one of the fastest observed fermentations, yet least energy e cient extractions found in nature. Furthermore it is the only known micro-organism to use the Entner-Doudoro (2-keto-3-deoxy-6- phosphogluconate) pathway anaerobically. The low energy yield of fermentation in Z. mobilis is a result of the usage of the Entner-Doudoro glycolytic pathway, which has half the energy yield per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway. The work presented in this thesis forms part of a larger project to compare glycolytic regulation in di erent micro-organisms Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus lactis. These organisms were chosen based on their usage of di erent glycolytic mechanisms. Kinetic models are suitable tools to draw a comparison between these organisms. The emphasis here is on the construction of a kinetic model of the Entner-Doudoroff glycolytic pathway as it occurs in Z. mobilis. The aim of this thesis was to characterise as many of the Entner-Doudoro pathway enzymes as possible, under standard conditions. This was done using enzyme assays, to obtain the kinetic parameters of each of the enzymes. Microtitre plate assays were used to characterise most of the enzymes of the Entner-Doudoro pathway. However, not all characterisations could be done using plate assay methods, as some intermediates were not commercially available to perform coupled assays. Nuclear magnetic resonance (NMR) spectroscopy was used to characterise these enzymes. These experimentally obtained parameters were then incorporated in a mathematical framework. Time simulations on the initial model were unable to reach a steady-state, with a build up of metabolic intermediates. A secondary model was constructed (using calculated maximal activities) which allowed us to identify discrepancies in the initial model. This showed that the experimentally determined maximal activities of three enzymes in lower glycolysis were unrealistically low, which might be due to protein denaturation by sonication. A nal model was constructed which incorporated a correction factor for these three enzymes. The models' predicted output (steady-state concentrations and ux) was compared to that of either literature or experimentally determined values, as a method to validate the model. The model output compared well to literature values. The constructed and partially validated kinetic model was then used as an analytical tool to identify points of control and regulation of glycolysis in Z. mobilis. The model presented in this work was also compared to published models. Our model relies much less on literature obtained values, and uses kinetic parameters experimentally determined under the same conditions. The parameters of the published models were obtained from the literature and in many instances, the assay conditions for these parameters were set-up to yield the maximum activity under non-physiological conditions. Furthermore, the number of excluded or assumed parameters is much less in our model. However, introduction of a milder, more predictable extraction technique for preparing cell lysates, should be considered for future work, to obtain the parameters that was not determined during this study. The published models do include reactions not included in our model (e.g ATP metabolism), which should be considered for inclusion, as we strive to construct a detailed kinetic model of glycolysis in Z. mobilis in the future. / AFRIKAANSE OPSOMMING: Sellul^ere metaboliese netwerke is komplekse stelsels, omdat hulle bestaan uit talle komponente met verskeie nie-lineêre interaksies. Om die funksionering van hierdie netwerke te verstaan, word hulle dikwels in hanteerbare stukke verdeel en individueel bestudeer. 'n Enkele komponent is egter nie in staat om die komplekse eienskappe van sulke stelsels te verklaar nie. Die veld van sisteembiologie het ontwikkel met die doel om sulke stelsels te bestudeer. Sisteembiologie maak gebruik van rekenaarmodelle as 'n metode om aspekte van lewende sisteme te beskryf. Kinetiese modelle van metaboliese paaie word gebou en gebruik as gereedskap om die biologiese stelsels te bestudeer en 'n kwantitatiewe beskrywing te bekom. Hierdie tesis beskryf die kwantitatiewe ontleding van 'n bakterie deur middel van 'n sisteembiologiese benadering. Zymomonas Mobilis is 'n staafvormige, Gram-negatiewe, nie-mobiele fakultatiewe ana erobe, en het een van die vinnigste waargenome fermentasies, maar met die minste energie-doeltre ende ekstraksie wat in die natuur aangetref word. Verder is dit die enigste bekende mikro-organisme wat die Entner-Doudoro (2-keto-3-dioksi-6-fosfoglukonaat) pad ana erobies gebruik. Die lae-energieopbrengs van fermentasie in Z. mobilis is 'n gevolg van die gebruik van die Entner-Doudoro metaboliese pad, wat die helfte van die energie-opbrengs per mol substraat lewer, in vergelyking met die bekende Embden-Meyerhof-Parnas pad. Die werk wat in hierdie tesis aangebied word, vorm deel van 'n groter projek om glikolitiese regulering in verskillende mikro-organismes te vergelyk, naamlik Z. mobilis, Escherichia coli, Sac- charomyces en Lactococcus lactis. Hierdie organismes is gekies op grond van hul gebruik van verskillende glikolitiese meganismes. Kinetiese modellering is 'n handige metode om 'n vergelyking tussen hierdie organismes te trek. Hierdie werk fokus op die bou van 'n kinetiese model van die Entner-Doudoro glikolitiese metaboliese pad soos dit in Z. mobilis voorkom. Die doel van hierdie tesis was om so veel moontlik van die Entner-Doudoro ensieme onder standaard-toestande te karakteriseer. Die kinetiese parameters van elk van die ensieme is met behulp van ensimatiese essai's bepaal. Vir die meeste essai's is 96-put mikrotiterplate gebruik, maar nie al die karakteriserings kon met behulp van hierdie metode gedoen word nie, omdat sommige intermediate nie kommersieel beskikbaar was om gekoppelde essai's mee uit te voer nie. Kernmagnetiese resonansie (KMR) spektroskopie is gebruik om hierdie ensieme te karakteriseer. Die eksperimenteel bepaalde parameters is opgeneem in 'n wiskundige raamwerk. Tydsimulasies op die aanvanklike model was nie in staat om 'n bestendige toestand te bereik nie, omdat metaboliete opgebou het. 'n Sekond^ere model is gebou (met behulp van berekende maksimale aktiwiteite) wat ons toegelaat om teenstrydighede in die aanvanklike model te identi seer. Dit het getoon dat die eksperimenteel bepaalde maksimale aktiwiteite van drie ensieme in die laer gedeelte van glikolise te laag was, waarskynlik as gevolg van prote en denaturering tydens die ultrasoniese disintegrasieproses. 'n Finale model is gebou waarin 'n korreksiefaktor vir hierdie drie ensieme opgeneem is. Die modelle se voorspelde uitset (bestendige toestand konsentrasies en uksie) is vergelyk met waardes uit die literatuur of wat ons self bepaal het, as 'n metode om die model te valideer. Die model uitset was in goeie ooreenstemming met hierdie waardes. Die gedeeltelik gevalideerde kinetiese model is voorts gebruik as 'n analitiese instrument om beheer en regulering van glikolise in Z. mobilis te ondersoek. Die model wat in hierdie werk ontwikkel is, is ook vergelyk met die vorige gepubliseerde modelle. Ons model berus baie minder op waardes uit die wetenskaplike literatuur, en maak gebruik van parameters wat eksperimenteel bepaal is, onder identiese toestande. Die parameters van die gepubliseerde modelle is meesal verkry uit die literatuur, en in baie gevalle was die eksperimentele kondisies vir hierdie analises opgestel om die maksimale aktiwiteit te lewer onder nie- siologiese toestande. Verder bevat ons model minder parameters wat of uitgesluit is of wie se waardes aangeneem moes word. In toekomstige werk sal daar egter klem gel^e moet word op 'n minder wisselvallige ekstraksietegniek vir die verkryging van selekstrakte, om sodoende parameters te identi seer wat nie in hierdie werk bepaal kon word nie. Die gepubliseerde modelle sluit ook reaksies in wat nie ingesluit is in ons model nie (bv. ATP metabolisme). Hierdie sou in ag geneem moet word vir insluiting in 'n toekomstige uitgebreide model, om daarna te streef om 'n gedetailleerde kinetiese model van glikolise in Z. mobilis te bou.

Polyuronic acids are an important constituent of seaweed and plants, and therefore represent a significant part of global biomass, providing an abundant carbon source for both terrestrial and marine heterotrophic bacteria. Through the action of polysaccharide lyases, polyuronic acids are degraded into unsaturated monouronic acid units, which are fed into the Entner-Doudoroff pathway where they are converted into pyruvate and glyceraldehyde-3-phosphate. The first step of this pathway was thought to occur non- enzymatically. A highly conserved sequence, kdgF was found in alginate and pectin utilization loci in a diverse range of prokaryotes, in proximity to well established enzymes catalyzing steps downstream in the Entner-Doudoroff pathway and I hypothesized that KdgF was involved in the catalysis of the first step of this pathway. The kdgF genes from both Yersinia enterocolitica and a locally acquired Halomonas sp. were expressed in Escherichia coli and their activity was examined using unsaturated galacturonic acid depletion activity assays. To gain perspective on the general structure of KdgF, x-ray crystallography was used to obtain a crystal structure of both HaKdgF and YeKdgF. These crystal structures provided insight into the molecular details of catalysis by the KdgF proteins, including their putative catalytic residues and a coordinated metal binding site for substrate recognition. To elucidate amino acids that may be involved in binding and/or catalysis, mutants were created in HaKdgF, and lack of activity was observed in four mutants (Asp102A, Phe104A, Arg108A, and Gln55A). The research done in this study suggests that KdgF proteins use a metal binding site coordinated by three histidines and several additional residues to cause a change in monouronic acid, thereby, affecting the unsaturated double bond. This suggests that KdgF is involved in the first step in the Entner-Doudoroff pathway, which is the linearization of unsaturated monouronic acids. / Graduate

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