The creation of enzymes displaying desired substrate specificity is an important objective of enzyme engineering. To help achieve this goal, computational protein design (CPD) can be used to identify sequences that can fulfill interactions required to productively bind a desired substrate. Standard CPD protocols find optimal sequences in the context of a single state, for example an enzyme structure with a single substrate bound at its active site. However, many enzymes catalyze reactions requiring them to bind multiple substrates during successive steps of the catalytic cycle. The design of multi-substrate enzyme specificity requires the ability to evaluate sequences in the context of multiple substrate-bound states because mutations designed to enhance activity for one substrate may be detrimental to the binding of a second substrate. Additionally, many enzymes undergo conformational changes throughout their catalytic cycle and the equilibrium between these conformations can have an impact on their substrate specificity. In this thesis, I present the development and implementation of two multistate computational protein design methodologies for the redesign of multi-substrate enzyme specificity and the modulation of enzyme conformational equilibrium. Overall, our approaches open the door to the design of multi-substrate enzymes displaying tailored specificity for any biocatalytic application.

<p>The Muoio lab studies the interplay between lipid whole body energy balance,</p><p>mitochondrial function and insulin action in skeletal muscle. Data from our lab suggests that lipid-induced insulin resistance in skeletal muscle may stem from excessive incomplete oxidation of fatty acids, which occurs when high rates of &beta;-­oxidation exceed TCA cycle flux (Koves et al., 2005; Koves et al., 2008). Most notably, we have shown that mice with a genetically engineered decrease in mitochondrial uptake and oxidation of fatty acids are protected against diet-­induced insulin resistance (Koves et al., 2008). This</p><p>suggests that an excessive and/or inappropriate metabolic burden on muscle</p><p>mitochondria provokes insulin resistance. Our working model predicts that: 1) high rates of incomplete &beta;-oxidation reflect a state of &rdquo;mitochondrial stress,&rdquo; and 2) that energy-overloaded mitochondria generate a yet unidentified signal that mediates insulin</p><p>resistance. One possibility is that this putative mitochondrial-derived signal stems from redox imbalance and disruptions in redox sensitive signaling cascades. Therefore, we are interested in identifying molecules that link redox balance, mitochondrial function and insulin action in skeletal muscle. The work described herein identifies thioredoxin-interacting protein (TXNIP) as an attractive candidate that regulates both glucose homeostasis and mitochondrial fuel selection.</p><p>TXNIP is a redox sensitive, &alpha;-arrestin protein that has been implicated as a negative regulator of glucose control. Mounting evidence suggested that TXNIP might play a key role in regulating mitochondrial function; however, the molecular nature of this relationship was poorly defined. Previous studies in TXNIP knockout mice reported that deficiency of this protein compromises oxidative metabolism, increases glycolytic activity and promotes production of reactive oxygen species (ROS), while also affording protection against insulin resistance. Therefore, we hypothesized that TXNIP might serve as a nutrient sensor that couples cellular redox status to the adjustments in mitochondrial function. We tested this hypothesis by exploiting loss of function models to evaluate the effects of TXNIP deficiency on mitochondrial metabolism and respiratory function.</p><p>In chapter 3, we comprehensively evaluated oxidative metabolism, substrate</p><p>selection, respiratory kinetics and redox balance in mice with total body and skeletal muscle-­specific TXNIP deficiency. Targeted metabolomics, comprehensive bioenergetics analysis, whole-body respirometry and conventional biochemistry showed that TXNIP deficiency results in reduced exercise tolerance with marked impairments in skeletal muscle oxidative metabolism. The deficits in substrate oxidation were not secondary to decreased mitochondrial mass or increased H₂O₂ emitting potential from the electron transport chain. Instead, the activities of several mitochondrial dehydrogenases involved in branched-chain amino acid and ketone catabolism, the tricarboxylic acid (TCA) cycle and fatty acid &beta;-oxidation were significantly diminished in TXNIP null muscles. These deficits in mitochondrial enzyme activities were accompanied by decreased protein abundance without changes in mRNA expression. Taken together, these results suggest that in skeletal muscle TXNIP plays an essential role in maintaining protein synthesis and/or stability of a subset of mitochondrial dehydrogenase enzymes that permit muscle use of alternate fuels under conditions of glucose deprivation.</p><p>Based on these conclusions, we questioned whether additional regulatory</p><p>mechanisms could contribute to the reduced oxidative metabolism in the absence ofTXNIP. Several metabolic enzymes of the TCA cycle have been shown to be redox-sensitive protein targets regulated by the thioredoxin (TRX1/TRX2) and glutathione (GSH) redox-mediated circuits. TXNIP has been shown to respond to oxidative stress by shuttling to the mitochondria where it binds to TRX2 and/or other proteins, thus affecting downstream signaling pathways, such as the apoptotic cascade. Therefore, we speculated whether there was a role for redox imbalance in mediating the mitochondrial phenotype of the TXNIP knockout (TKO) mice. In chapter 4, we present preliminary evidence that increased glucose uptake promotes non-mitochondrial ROS production, causing a shift in redox balance, decreased GSH/GSSG, and S-glutathionylation of &alpha;-­ketoglutarate dehydrogenase (&alpha-KGD). This post-translational modification protects the protein from permanent oxidative damage, but at the cost of reversible loss of activity and subsequent disruption of TCA cycle flux that contributes, in part, to the diminished oxidative metabolism observed in the TXNIP deficient mice.</p><p>In aggregate, this work sheds new light onto the physiological role of TXNIP in</p><p>skeletal muscle as it pertains to substrate metabolism and fuel switching in response to nutrient availability. This work has important implications for metabolic diseases such as obesity and type 2 diabetes, which are characterized by marked disruptions in fuel selection.</p> / Dissertation

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway. / AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg.

Made available in DSpace on 2017-07-10T17:48:26Z (GMT). No. of bitstreams: 1 Doglas_Lazzeri.pdf: 319692 bytes, checksum: ab1f14cee65939d583d1aaf7427d892d (MD5) Previous issue date: 2011-07-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aiming to determine the digestible isoleucine requirement for barrows from 15 to 30 kg, two experiments were conducted, being one of digestibility and another of performance. Ten crossbreed barrows with average initial weight of 15.00 ± 0.27 kg were used to determine the ileal digestibility coefficient of amino acids from basal diet. The animals were housed individually in metabolic cages in a completely randomized design, with two treatments, five replicates and one animal per experimental unit. The treatments consisted of a basal diet with 14.13% crude protein and 0.45% isoleucine, and a free protein diet. Forty crossbreed barrows with average initial weight of 15.00 ± 0.87 kg were used to determine the digestible isoleucine requirement. The animals were distributed in a randomized block design with five treatments (0.45, 0.52, 0.59, 0.66 and 0.73% digestible isoleucine), four replicates and two animals per experimental unit. The coefficients of true ileal digestibility of essential amino acids were higher than the coefficients of apparent ileal digestibility and threonine, methionine+cystine and valine had the biggest increases. The true ileal digestibility of basal diet for lysine, threonine and tryptophan were higher than the true ileal digestible calculated amino acids. The same was not observed for true ileal digestibility of basal diet of arginine, histidine, isoleucine, leucine, methionine, methionine+cystine, phenylalanine and valine. The protein deposition rate had a quadratic effect (P<0.05) which the level of 0.600% digestible isoleucine had the highest protein deposition in carcasses of pigs. The daily isoleucine intake had quadratic effect (P<0.01), which level of 0.938% digestible isoleucine provided the higher intake. There was linear effect (P<0.01) on efficiency of isoleucine utilization for weight gain, with reduction on values of EIleUWG as levels of digestible isoleucine increased in rations. A quadratic effect (P=0.09) of digestible isoleucine levels on the final weight was observed, with better values for the level 0.599%. The level of 0.60% digestible isoleucine obtained in the present work provided a digestible isoleucine:lysine relation of 0.60. The daily requirement of digestible isoleucine was 5.86 g/day, providing 1.76g of digestible isoleucine/Mcal of metabolizable energy / Com o objetivo de determinar a exigência de isoleucina digestível para suínos machos castrados dos 15 aos 30 kg de peso vivo, foram realizados dois experimentos, um de digestibilidade e outro de desempenho. Para determinar o coeficiente de digestibilidade ileal dos aminoácidos da ração basal, foram utilizados 10 suínos, machos castrados, mestiços, com peso médio inicial de 15,00±0,27 kg, alojados individualmente em gaiolas de metabolismo, em um delineamento inteiramente ao acaso, com dois tratamentos, cinco repetições e um animal por unidade experimental. Os tratamentos consistiram de uma dieta basal, com 14,13% de proteína bruta e 0,450% de isoleucina, e uma dieta isenta de proteína. Na determinação da exigência de isoleucina digestível para suínos machos castrados, foram utilizados 40 suínos mestiços, com peso médio inicial de 15,00±0,87 kg, distribuídos em um delineamento experimental de blocos casualizados, com cinco tratamentos (0,45; 0,52; 0,59; 0,66 e 0,73% de isoleucina digestível), quatro repetições e dois animais por unidade experimental. Os valores dos coeficientes de digestibilidade ileal verdadeira dos aminoácidos essenciais foram maiores do que os coeficientes de digestibilidade ileal aparente, sendo a treonina, a metionina + cistina e a valina os aminoácidos que apresentaram os maiores aumentos. Os valores de digestibilidade ileal verdadeira da dieta basal para lisina, treonina e triptofano foram superiores aos valores de aminoácidos digestíveis ileais verdadeiros calculados. O mesmo não foi observado para os valores de digestibilidade ileal verdadeira da dieta basal dos aminoácidos arginina, histidina, isoleucina, leucina, metionina, metionina + cistina, fenilalanina e valina. A taxa de deposição de proteína apresentou efeito quadrático (P<0,05), em que o nível de 0,600% de isoleucina digestível proporcionou a maior deposição de proteína na carcaça dos suínos. Houve efeito quadrático (P<0,01) dos níveis de isoleucina digestível sobre o consumo diário de isoleucina, com estimativas de melhor consumo para o nível de 0,938%. Foi observada diferença linear (P<0,01) apenas para a eficiência de utilização de isoleucina para ganho, em que o modelo linear apresentou redução nos valores de eficiência à medida que aumentaram os níveis de isoleucina digestível nas rações. Foi observado efeito quadrático (P=0,09) dos níveis de isoleucina digestível sobre o peso final, com melhores valores obtidos para o nível de 0,599%. O nível de 0,60% de isoleucina digestível, obtido no presente estudo, proporcionou uma relação isoleucina:lisina digestível de 0,60. A exigência diária de isoleucina digestível foi de 5,86 g/dia, proporcionando um consumo de 1,76 g de isoleucina digestível/Mcal de energia metabolizável