<p>Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3‑butanediol, a product formed in an alternative pathway to mixed acid fermentation.</p> <p>While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism. </p> <p>Here, the ALS and AHAS enzymes from <i>Klebsiella pneumoniae</i> were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site‑saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS <i>in vivo</i>.</p>

Sucrose synthase (Susy) is a key enzyme of sucrose metabolism, catalysing the reversible conversion of sucrose and UDP to UDP-glucose and fructose. Therefore, its activity, localization and function have been studied in various plant species. It has been shown that Susy can play a role in supplying energy in companion cells for phloem loading (Fu and Park, 1995), provides substrates for starch synthesis (Zrenner et al., 1995), and supplies UDP-glucose for cell wall synthesis (Haigler et al., 2001). Analysis of the Arabidopsis genome identifies six Susy isoforms. The expression of these isoforms was investigated using promoter-reporter gene constructs (GUS) and real time RT-PCR. Although these isoforms are closely related at the protein level they have radically different spatial and temporal patterns of expression in the plant with no two isoforms showing the same distribution. More than one isoform is expressed in all organs examined. Some of them have high but specific expression in particular organs or developmental stages whilst others are constantly expressed throughout the whole plant and across various stages of development. The in planta function of the six Susy isoforms were explored through analysis of T-DNA insertion mutants and RNAi lines. Plants without the expression of individual isoforms show no differences in growth and development, and are not significantly different from wild type plants in soluble sugars, starch and cellulose contents under all growth conditions investigated. Analysis of T-DNA insertion mutant lacking Sus3 isoform that was exclusively expressed in stomata cells only had a minor influence on guard cell osmoregulation and/or bioenergetics. Although none of the sucrose synthases appear to be essential for normal growth under our standard growth conditions, they may be necessary for growth under stress conditions. Different isoforms of sucrose synthase respond differently to various abiotic stresses. It has been shown that oxygen deprivation up regulates Sus1 and Sus4 and increases total Susy activity. However, the analysis of the plants with reduced expression of both Sus1 and Sus4 revealed no obvious effects on plant performance under oxygen deprivation. Low temperature up regulates Sus1 expression but the loss of this isoform has no effect on the freezing tolerance of non acclimated and cold acclimated plants. These data provide a comprehensive overview of the expression of this gene family which supports some of the previously reported roles for Susy and indicates the involvement of specific isoforms in metabolism and/or signalling. / Saccharose spielt eine zentrale Rolle in höheren Pflanzen. Es zählt zu den wichtigsten Kohlenhydraten und wird als Nährstoff, Speicherstoff (z.B. in Zuckerrüben, Zuckerrohr, Mohrrüben) oder auch als potentielles Signalmolekül verwendet. Saccharose ist eines der primären Endprodukte der Photosynthese in den grünen Blättern der Pflanzen, kann aber auch in nicht-photosynthetisch aktiven Geweben (z.B. in keimenden Samen) synthetisiert und verstoffwechselt werden. Die Saccharosesynthase (Susy) stellt ein Schlüsselenzym im Saccharosestoffwechsel dar. Es katalysiert die reversible Umwandlung von Saccharose zu UDP-Glukose und Fruktose. Die Aktivität, die Lokalisierung und die Funktionen der Susy wurden bereits in verschiedenen Pflanzenarten untersucht. Dabei hatte sich herausgestellt, daß die Susy eine wichtige Rolle in der Bereitstellung von Energie für Transportprozesse spielt. Außerdem stellt Susy die Substrate für die Stärkesynthese in Speichergeweben, sowie fast alle Substrate für die Zellwandsynthese bereit. Eine Untersuchung des Genoms von Arabidopsis thaliana ergab, daß die Ackerschmalwand sechs Isoformen der Susy besitzt. Die Expression dieser Isoformen wurde mittels Echtzeit RT-PCR analysiert. Obwohl die verschiedenen Isoformen auf Proteinebene in ihrer Sequenz sehr ähnlich sind, zeigen sie Unterschiede in ihrem zeitlichen und räumlichen Auftreten innerhalb der Pflanze. Einige der Isoformen sind hoch exprimiert in speziellen Organen oder Entwicklungsstufen der Pflanze. Andere hingegen sind gleichmäßig in der ganzen Pflanze und über verschiedene Entwicklungsstufen hinaus exprimiert. In allen untersuchten Organen der Pflanze ist mehr als eine Isoform exprimiert. Um die spezifische Funktion der einzelnen Isoformen aufzuklären, wurden für alle sechs Saccharosesynthasen Mutanten-Linien isoliert und analysiert. Alle Pflanzen, bei denen die Expression einer bestimmten Isoform fehlte, zeigten im Vergleich zu Wildtyppflanzen keine signifikanten Unterschiede in Wachstum und Entwicklung. Des Weiteren waren die Gehalte an Stärke, Saccharose und Zellulose in Blättern und Wurzeln im Vergleich zu Wildtyppflanzen unverändert. Mutanten, denen die ausschließlich in Schließzellen lokalisierte Isoform Sus3 fehlte, zeigten nur geringe Veränderungen in der Osmoregulation und/oder der Bioenergetik der Schließzellen. Daraus kann gefolgert werden, dass in dem Ackerunkraut Arabidopsis keine der Saccharosesynthasen essentiell für normales Wachstum unter Standardbedingungen ist. Es ist jedoch möglich, dass Saccharosesynthasen unter Stressbedingungen benötigt werden. Es war bereits bekannt, dass einzelne Isoformen der Susy auf Stress reagieren und in ihrer Expression verändert sind. Es konnte gezeigt werden, daß Sauerstoffmangel zu einer Erhöhung der Expression der Isoformen Sus1 und Sus4 und zu einer Zunahme der Susy Gesamtaktivität führt. Die Analyse von Pflanzen mit reduzierter Expression von Sus1 und Sus4 zeigte jedoch, dass Sauerstoffmangel keinen offensichtlichen Einfluss auf das Wachstum dieser Pflanzen hat. Niedrige Temperaturen führen zu einer Erhöhung der Sus1 Expression, aber auch ein Verlust dieser Isoform hat keinen Einfluss auf die Gefriertoleranz von normalen oder an Kälte akklimatisierten Pflanzen. Diese Ergebnisse bieten einen umfassenden Einblick in die Expression der Genfamilie der Saccharosesynthase; sie untermauern die genannten Funktionen der Saccharosesynthase und weisen auf eine mögliche Beteiligung mehrerer Isoformen am Saccharosestoffwechsel und/oder der Signaltransduktion hin.

Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xii, 73 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 64-70).

The enzyme α-isopropylmalate synthase (IPMS) catalyses the reaction between acetyl coenzyme A (AcCoA) and α-ketoisovalerate (KIV) to produce free coenzyme A and α isopropylmalate (IPM). This reaction is a key control point in the biosynthesis of a leucine, a pathway absent in animals but present in plants, fungi and bacteria. As a result, IPMS is a antibiotic and herbicidal target that has been validated by knockout studies for M. tuberculosis, the causative agent of tuberculosis. Engineered IPMSs have also been used in the fermentative production of long chain alcohols for use as fuels. IPMS belongs to a family of related enzymes called α-ketoacid: AcCoA re-aldolases (KARAs), with each subfamily differing in the specific α-ketoacid that AcCoA is reacted with. The known KARA subfamilies are IPMS, citramalate synthases (CMSs), homocitrate synthases (HCSs), methylthioalkylmalate synthases (MAMSs) and re-citrate synthases (RCSs), respectively involved in the biosynthesis of isoleucine, lysine, glucosinolates and TCA cycle intermediates. This thesis describes work aimed at improving understanding of both specific subfamilies of KARA enzymes and also the genetic and functional relationships between the subfamilies. A particular emphasis is placed on relating primary structure to function, allowing the inference of function from a very small subset of residues. IPMSs are divided into two classes, the Mtu-like IPMSs and the much less studied Eco-like IPMSs. Chapter 2 details the expression and characterisation of the Eco like IPMS from N. meningitidis (NmeIPMS). Overall NmeIPMS showed similar properties to MtuIPMS, but unlike that enzyme NmeIPMS is inhibited by high divalent metal ion concentrations, does not require monovalent metal ions, and shows some activity with the α-ketoacid 3-methyl α ketovalerate. Several previous results showing inhibitory activity of Zn2+, Cd2+ and bromopyruvate were also found to be the results of interference with the assay system and all three were found to be much weaker inhibitors than previously determined.   Phylogenetic analysis of the different KARA subfamilies revealed certain specific positions that were believed to control substrate specificity. Chapter 3 details mutagenesis experiments on MtuIPMS that probe the function of these residues. Once the importance of the residues had been established, substitutions were made in which IPMS residues were replaced with their equivalents from HCSs and CMSs in order to change substrate specificity. The most successful result was the Y169L substitution based on HCS, which decreased the specificity constant with KIV by four orders of magnitude while improving other activities, successfully shifting the best activity to the unbranched α-ketoacid α-ketobutyrate. Chapter 4 of this thesis details the purification and functional testing of the RCS from C. saccharolyticus (CscRCS), the first thermophilic RCS characterised. CscRCS was found to have an extremely low Km for its substrate oxaloacetate (1.7 µM), believed to be an adaptation to the instability of oxaloacetate at the temperatures CscRCS operates at in vivo. The enzyme also showed competitive affinity by α-ketoglutarate, the end product of the pathway. Unlike other characterised RCSs, CscRCS showed no oxygen sensitivity. The phylogenetic analysis conducted for this thesis identified a subfamily of KARAs dubbed pseudo-IPMSs (PIPMSs) that showed no substantial homology to any studied subfamily. In Chapter 5 the PIPMS from T. thermophilus (TthPIPMS) was expressed and characterised. TthPIPMS showed many features of a CMS, being most active with the same substrate (pyruvate) and sensitive to the same inhibitor (isoleucine). Unlike the previously studied CMS subfamilies, TthPIPMS possesses a nanomolar IC50 for its inhibitor, and also shows substantial activity as an RCS. The results of these chapters are then drawn together in Chapter 6 to create a picture of the relationships between the KARA enzymes, in terms of their functional characteristics as well as the sequence and evolutionary relationships between them that have bought about their diverse functions.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains xi, 78 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 59-67).

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains xvi, 184 p. : ill. Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xi, 172 p. : ill. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 132-150). Also available on the Internet.