The purpose of this research was to characterize the Escherichia coli, E. coli, allantoinase enzyme. Allantoinase catalyses the conversion allantoin to allantoate via the hydrolysis of a cyclic amide bond and is coded for by the allB gene. The enzyme is a member of the amidohydrolase superfamily. Amidohydrolase superfamily enzymes have a common (αβ)8-barrel structure but catalyze the hydrolysis of many different substrates by a common mechanism. The structural characteristics and roles of divalent cations of enzymes in this superfamily will be discussed and related to previous work conducted on allantoinases. In this work, the metal dependence of allantoinase was initially studied by Mn, Co, Zn, Cd, and Ni-supplemented assays of enzyme of very low metal content. By changing the growth conditions under which the allB was overexpressed in E. coli, and the addition of Zn, Co or Mn to the culture, enzyme with bound Zn (ZnALN), Co (CoALN) or Mn (MnALN) was produced. The pH dependence of log (kcat/KM) for allantoinase in the presence of MnCl2, ZnALN and CoALN followed a bell-shaped curve, indicating that one ionizable group needed to be deprotonated and the deprotonation of a second group caused a decrease in catalytic activity. The pK1 for ionization at low pH was dependent upon which divalent cation was present and is concluded to be that of the deprotonation of water. A structural model of allantoinase with bound allantoin was constructed and used to determine which amino acid residues may be involved in catalysis. Allantoinase mutants R67K, C152A, C152S, C287A, C287S, S317A, D315N and W332F were purified. The kinetic parameters kcat, KM and kcat/KM of wild type and mutant allantoinases were compared. The possible roles of these amino acid residues in catalysis and substrate binding, and the results of the pH rate profiles are discussed. A catalytic mechanism for allantoinase is proposed.

Thesis (MSc Food Science)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: South Africa is a water scarce country and farmers are forced to irrigate crops with river water. Contamination of South African rivers has been reported and the carry-over of bacteria from river water to produce has been confirmed. Foodborne outbreaks linked to fresh produce are increasing world-wide. A total of 151 fresh produce samples (lettuce, tomatoes, beans, peas, coriander, basil, mint, rocket, thyme, spinach, cabbage, parsley and sprouts) were sourced from small-scale and commercial farms, farmers’ markets and retail outlets. Total coliforms (TC) and E. coli loads on the produce were determined with Colilert-18. Isolates were phenotypically characterised and identified with the API system and the E. coli identification confirmed with uidA PCR. Sixty-three E. coli isolates were identified. Three were not identified as E. coli with the API system but were positive for the uidA gene. The TC loads for the produce from the farms, farmers’ markets and retail outlets were all in the range of log 3 to log 8.38 MPN.100 mL-1. Escherichia coli was found to be most prevalent on produce samples from farmers’ markets with the highest E. coli load (log 7.38 MPN.100 mL-1) on cabbage sampled from a commercial farm. Escherichia coli were present on 8% of the produce samples. The maximum TC and E. coli loads found on the fresh produce were log 8.38 and log 7.38 MPN.100 mL-1, respectively. The lowest risk in terms of TC and E. coli presence and load was observed on fresh produce from retail outlets and the highest risk was on fresh produce from farmers’ markets. Phenotypic dendrograms and a PCA plot were statistically constructed to determine similarity groupings of the isolates and three main E. coli clusters were formed. These three clusters could not be directly linked to a specific produce type or source type. A larger variation E. coli phenotypes was observed present on fresh produce within the three clusters. All E. coli isolates were also subjected to triplex and multiplex PCR analysis to identify their phylogenetic groups and the presence of INPEC and ExPEC strains. Fourteen isolates belonged to genotypic group A0, 11 to A1, 20 to B1, 7 to B23 and 11 to D2. Thus a large variation E. coli genotypes are present but it cannot be linked to a specific source type or produce type. Multiplex PCR testing for INPEC revealed that none of the E. coli isolates were carriers of the INPEC genes. The isolates were also tested for the presence of ExPEC gene sequences: papA, papC, sfa/foc, iutA, kpsMT II and afa/dra. None of the isolates were classified as ExPEC (which required the presence of two or more genes) but three of the isolates did test positive for the presence of the kpsMT II gene. The latter could indicate that potentially pathogenic E. coli can be evolving in the environment and increase the risk of pathogenic E. coli occurring on fresh produce. In conclusion, the presence of E. coli (commensal or pathogenic) on fresh produce is unacceptable according the South African Department of Health. According to this study the identification of E. coli types could not be correlated with the presence of E. coli on the different produce types and thus the presence of E. coli on fresh produce is unpredictable. It is recommended that extensive safety precautions should be in place throughout every step in the production chain from harvest to the consumer’s kitchen to reduce the probability of contamination of fresh produce. / AFRIKAANSE OPSOMMING: Suid-Afrika is ‘n waterskaars land en boere word gedwing om rivier water te gebruik vir gewas besproeiing. Kontaminasie van Suid-Afrikaanse riviere is al telkemale aangemeld en die oordrag van bakterieë vanaf rivierwater na vars produkte is al voorheen bevestig. Voedselverwante uitbrake wat gekoppel is aan vars produkte is besig om wêreldwyd toe te neem. ‘n Totaal van 151 vars produk monsters (blaarslaai, tamaties, boontjies, ertjies, koljander, basilie, kruisement, roket, tiemie, spinasie, kool, pietersielie en spruite) was verkry van klein-skaalse en kommersiële plase, plaasmarkte en kettingwinkels. Totale kolivorme (TK) en E. coli tellings op die vars produkte is bepaal deur middel van Colilert-18. Isolate word fenotipies gekarakteriseer en geïdentifiseer met die API sisteem en die E. coli identifikasie is bevestig met uidA PKR. Drie-en-sestig E. coli isolate is geïdentifiseer. Drie is nie met met die API sisteem as E. coli geklassifiseer nie, maar was wel positief vir die uidA geen. Die TK tellings vir die vars produkte van die plase, plaasmarkte en kettingwinkels was almal in die reeks van log 3 tot log 8.38 MPN.100 mL-1. Escherichia coli teenwoordigheid was die meeste op groente monsters van plaasmarkte, maar die hoogste E. coli telling (log 7.83 MPN.100 mL-1) was op ‘n kool monster van ‘n kommersiële plaas. Escherichia coli was teenwoordig op 8% van die vars produk monsters. Die maksimum TK en E. coli wat teenwoordig was op die vars produkte was log 8.38 en log 7.38 MPN.100 mL-1 onderskeidelik. Die laagste risiko in terme van TK en E. coli teenwoordigheid en tellings is waargeneem op vars produkte van kettingwinkels en die hoogste risiko is op vars produkte van plaasmarkte. Fenotipiese dendrogramme en ‘n PKA plot is statisties gekonstrueer om ooreenstemende groepe van isolate te identifiseer en drie hoof groepe is gevorm. Daar kon geen direkte verband gevind word tussen hierdie drie groepe en ‘n spesifieke produk-tipe of ‘n spesifieke bron-tipe nie. ‘n Groter variasie in E. coli fenotipes teenwoordig op die vars produkte is waargeneem binne die drie groepe. Alle E. coli isolate was onderworpe aan tripleks en multipleks PKR analise om die filogenetiese groep van elke isolaat te bepaal en of enige INPEC of ExPEC stamme teenwoordig is. Veertien isolate behoort aan genotipiese groep A0, 11 aan A1, 20 aan B1, 7 aan B23 en 11 aan D2. Dus is ‘n groot variasie E. coli genotipes teenwoordig maar dit kan nie gekoppel word aan ‘n spesifieke produk-tipe of bron-tipe nie. Multipleks PKR analise vir INPEC het gewys dat geeneen van die E. coli isolate enige INPEC gene dra nie. Die isolate is ook getoets vir die teenwoordigheid van ExPEC geen volgordes: papA, papC, sfa/foc, iutA, kpsMT II en afa/dra. Geeneen van die isolate is geklassifiseer as ExPEC (wat die teenwoordigheid van twee of meer gene vereis) nie, maar drie van die isolate het wel positief getoets vir die teenwoordigheid van die kpsMT II geen. Laasgenoemde kan ‘n aanduiding wees dat potensiële patogeniese E. coli in die omgewing kan ontwikkel en dus dan die risiko van die teenwoordigheid van patogeniese E. coli op vars produkte sal verhoog. Ter afsluiting, die teenwoordigheid van E. coli (nie-patogenies en patogenies) op vars produkte is onaanvaarbaar volgens die Suid-Afrikaanse Departement van Gesondheid. Volgens hierdie studie kan die identifisering van E. coli tipes nie gekorreleer word met die teenwoordigheid van E. coli op verskillende produk-tipes nie en dus is die teenwoordigheid van E. coli op vars produkte onvoorspelbaar. Dit word aanbeveel dat ekstensiewe voorsorgmaatreëls in plek moet wees in elke stap dwarsdeur die produksie ketting, vanaf oestyd tot in die verbruiker se kombuis, om die moontlikheid van vars produk kontaminasie te verminder.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-02-17T18:26:18Z No. of bitstreams: 1 Maria Goreth Matos de Andrade Barberino... Distribuição clonal 2013.pdf: 2055064 bytes, checksum: da2e73dad9d911837f5434c00617e1b0 (MD5) / Made available in DSpace on 2014-02-17T18:26:18Z (GMT). No. of bitstreams: 1 Maria Goreth Matos de Andrade Barberino... Distribuição clonal 2013.pdf: 2055064 bytes, checksum: da2e73dad9d911837f5434c00617e1b0 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / A infecção do trato urinário (ITU) é considerada a segunda infecção mais comum em humanos, estima-se que ocorram cerca de 150 milhões de casos de ITUs por ano no mundo. O aumento das taxas de resistência aos antimicrobianos entre os uropatógenos tem tornado mais difícil o tratamento das ITUs. Objetivo: Determinar a distribuição clonal das cepas de E. coli isoladas em pacientes com ITU adquirida na comunidade de acordo com o perfil de susceptibilidade aos antimicrobianos e avaliar o papel dos grupos clonais na disseminação e persistência da resistência nestas infecções. Métodos: Foram isoladas 874 cepas de E. coli em pacientes com ITU, procedentes de unidades ambulatoriais em três hospitais na cidade de Salvador–Ba, no período de 2001 a 2009. O perfil de susceptibilidade foi determinado por microdiluição em placa (Microscan-Siemens®). Nas amostras selecionadas para genotipagem (n=275), a identificação dos grupos clonais foi realizada pela comparação dos padrões de PFGE, utilizando os critérios de Tenover (1995). Em todas as etapas do estudo foi utilizada como controle de qualidade a cepa ATCC E. coli 25922. Resultado: Entre os antibióticos testados, a maior prevalência de resistência foi encontrada para ampicilina (AMP) (49%), cefalotina (12-33%) e sulfametoxazol-trimetropin (SXT) (36-42%). A taxa de resistência à ciprofloxacina (CIP) variou de 9 a 14%. Na análise da distribuição clonal, segundo os fenótipos de resistência aos antimicrobianos, encontramos maior predomínio de um grupo clonal CgA (63%) entre as cepas consideradas multidroga resistentes. Diferentemente das amostras com algum grau de resistência ou multi-sensíveis, nas quais observamos diversidade clonal. Conclusão: A alta prevalência de resistência a SXT, AMP e cefalotina contraindica o uso destes antimicrobianos no tratamento empírico das ITU adquiridas na comunidade. A taxa de resistência à CIP relativamente alta, alerta para o aumento e disseminação de resistência a este antimicrobiano na comunidade. Isto irá dificultar e onerar o tratamento destas infecções. Observamos a surgimento de um grupo clonal (CgA) no período final do estudo (2008 a 2009) associado às cepas multidroga resistentes. Este achado sugere que a expansão de determinados clones pode ter um papel importante na disseminação de resistência bacteriana em ITUs adquiridas na comunidade. / Urinary tract infection (UTI) is considered the second most common infection in humans. It is estimated that there are about 150 million cases of UTIs per year worldwide. Increasing rates of antimicrobial resistance among uropathogens challenges UTI treatments. Objective: To determine the distribution of clonal strains of E. coli isolated from patients with community-acquired UTI according to the profile of antimicrobial susceptibility; and to evaluate the role of clonal groups in the spread and persistence of resistance in these infections. Methods: Eight hundred seventy four strains of E. coli were isolated from patients with UTI, coming from outpatient clinics in three hospitals in the city of Salvador - Bahia, from 2001 to 2009. The susceptibility profile was determined by broth microdilution method (Siemens - Microscan ®). The samples selected for genotyping (n = 275) were identified for clonal groups by comparing the patterns of PFGE, using the criteria of Tenover (1995). All study stages were quality control by strain E. coli ATCC 25922. Results: Among the antibiotics tested, the highest prevalence of resistance was for ampicillin (AMP) (49%) followed by trimethoprim - sulfamethoxazole (SXT) (36-42%) and for cephalothin (12-33%). The rate of resistance to ciprofloxacin (CIP) ranged between 9-14 %. In the Clonal Analysis distribution, performed according to antimicrobial resistance phenotypes, we found a higher prevalence of a clonal group CgA (63%) among multidrug resistant strains. This result differs from samples with some degree of resistance or multi-sensitive in which we observed clonal diversity. Conclusion: The high prevalence of resistance to SXT, AMP, and cephalothin contraindicate the use of these antibiotics in the empirical treatment of community-acquired UTI. The relatively high rate of resistance to CIP, raises attention to the increase and spread of antimicrobial resistance in this community and potentially complicate and encumber the treatment of these infections. We observe the emergence of a clonal group (CgA) in the final period of the study (2008-2009) associated with multidrug resistant strains. This finding suggests that the expansion of particular clones may have an important role in the spread of bacterial resistance in community-acquired UTI.

The growing problem of antibiotic resistance and the lack of promising prospective antibiotics have forced us to search for new classes of antibiotics. Among the candidates to develop into future antibacterials are antimicrobial peptides (AMPs). These potent, broad spectrum compounds are important components of innate immunity of organism from all kingdoms of life. One such family of mini-proteins from plants is called cyclotides, whose members are defines by cyclic backbone and a cystine knot (CCK), which confers to them extreme stability in the face of biological, chemical and physical insults.     Some cyclotides possess Gram-negative specific antibacterial activity; the purpose of this thesis was to characterize how these molecules kill bacteria, and how bacteria would respond to treatment with cyclotides. For this purpose, Salmonella enterica and Escherichia coli mutants resistant to the cyclotides cycloviolacin O2 and cycloviolacin O19, respectively, were selected. These mutants were characterized by whole genome sequencing, genetic reconstitution, fitness measurements, and cross-resistance studies. These studies identified a number of genetic pathways for resistance development to cyclotides. These mutants displayed variable fitness profiles in laboratory growth media and in mice competition experiments, with some mutants possessing a fitness advantage in mice. Cross-resistance studies resulted in the identification of several cases of cross-resistance and collateral sensitivity between cyclotides and other AMPs/antibiotics.      Antimicrobial effects of cyclotides were assayed in different conditions and in bacterial organisms with different surface characteristics. In addition, immunolocalization experiments were performed to explore the biological distribution of cyclotides in plants and to determine the mechanism of action of cyclotides in bacteria, respectively. Antibodies raised against cyO2 were used for this purpose. Immunohistochemical techniques applied to plant cells, tissues and organs provided the information that cyclotides were distributed in all plant organs, and were found in tissues vulnerable to pathogen attack, and that cyclotides were stored in the vacuoles of plant cells. Immunogold staining of cyclotide treated cells of S. typhimurium, showed effects of cyclotide treatment on the cell envelope components as well as cytoplasm. A higher number of cyclotide molecules was associated with the cell envelope, but a considerable fraction of them penetrated into the cytoplasm.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-10-31T15:34:23Z No. of bitstreams: 1 texto completo.pdf: 1129658 bytes, checksum: 7294d8748cb6a2c8b02db97c958b323f (MD5) / Made available in DSpace on 2016-10-31T15:34:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1129658 bytes, checksum: 7294d8748cb6a2c8b02db97c958b323f (MD5) Previous issue date: 2003-04-14 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Probióticos, na forma de produtos lácteos fermentados ou não, são conhecidos como alimentos funcionais e têm sido recomendados como adjunto dietético para indivíduos com hipercolesterolemia. Com o objetivo de avaliar seu efeito no metabolismo lipídico e em outras propriedades funcionais do tubo digestório foram conduzidos cinco ensaios biológicos, envolvendo 104 ratos e 48 coelhos. A modulação da colesterolemia foi avaliada em ratos que receberam dieta basal ou dieta hiperlipídica suplementada com diferentes células de L. acidophilus CH5, L. acidophilus NCFM e L. casei spp., por um período de 14 dias. Observou-se que as espécies L. acidophilus CH5 e L. casei spp. apresentaram praticamente o dobro da atividade da enzima BSH de L. acidophilus NCFM. A suplementação de probióticos aos animais não alterou (P>0,05) o consumo alimentar, o coeficiente de eficiência alimentar, o ganho de peso, o índice de peso de fígado, o índice de peso do baço, o índice aterogênico e a concentração de colesterol total, HDL-C, VLDL+LDL-C, ácidos biliares e triacilgliceróis entre os grupos experimentais. Por outro lado, a dieta hiperlipídica administrada aos animais não aumentou a concentração de colesterol sangüíneo (P>0,05). Avaliando-se o efeito da administração de diferentes concentrações de L. casei spp. (104, 106, 108 UFC/mL) em ratos normo e hipercolesterolêmicos, por um período de 14 dias, verificou-se que o ganho de peso (P<0,05), o coeficiente de eficiência alimentar (P<0,01) e os parâmetros sangüíneos (índice aterogênico e níveis séricos de colesterol total, triacilgliceróis, HDL-C, LDL+VLDL-C) (P<0,01) dos animais que receberam a dieta hiperlipídica foram superiores aos dos animais que receberam a dieta basal. No entanto, independentemente do tipo de dieta, as diferentes concentrações de L. casei spp. administradas aos animais não exerceram efeito sobre esses parâmetros (P>0,05). Embora houvesse aumento (P<0,01) no peso do fígado, na concentração de colesterol hepático e na concentração de colesterol e ácidos biliares fecais nos animais alimentados com dieta hiperlipídica, quando comparados com os que receberam a dieta basal. Observa-se que o efeito de dose foi significativo no colesterol hepático (P<0,01) e fecal (P<0,05), independentemente do tipo de dieta. No entanto, não houve diferença significativa (P>0,05) entre as diferentes concentrações de L. casei spp. Repetindo a experimentação anterior em coelhos normo e hipercolesterolêmicos, porém utilizando as concentrações 105, 107, 109 UFC/mL, verificou-se que os parâmetros sangüíneos (P<0,01), o peso do fígado (P<0,01) e a concentração de colesterol hepático e colesterol fecal (P<0,01) dos animais que receberam a dieta hiperlipídica foram superiores aos dos animais que receberam a dieta basal. No entanto, diferentemente de ratos, o efeito de dose não foi significativo (P>0,05) para todas as características analisadas. Quanto ao efeito de dose em outras propriedades funcionais do tubo digestório em ratos e coelhos normocolesterolêmicos, constatou-se que as diferentes concentrações probióticas testadas aumentaram os níveis de Lactobacillus (P<0,01) e reduziram os níveis de E. coli (P<0,05) e a atividade da enzima â-glucuronidase (P<0,01) em ratos, mas não em coelhos (P>0,05). Por outro lado, não houve alteração no peso do baço, nem na contagem bacteriana neste órgão (P>0,05), mas houve aumento do comprimento das vilosidades intestinais (P<0,01) nos dois modelos animais. Portanto, conclui- se que, embora não houvesse alteração na colesterolemia dos animais, a suplementação de doses crescentes de L. casei spp. exerceu alguma influência no metabolismo de colesterol, na morfologia intestinal e na modulação da microbiota intestinal, aumentando os níveis de lactobacilos e reduzindo os níveis de E. coli, dos dois modelos estudados. / Probiotics, whether in the form of fermented dairy products or not, are known as functional foods and have been recommended as dietary adjuncts to individuals with hypercholesterolemia. Five bioassays were carried out to evaluate the effect of probiotics on the lipid metabolism and other functional properties of the alimentary canal in 104 rats and 48 rabbits. Cholesterolemia modulation was evaluated in rats fed a basal diet or hyperlipidic diet supplemented with different cells of L. acidophilus CH5, L. acidophilus NCFM and L. casei spp. for 14 days. The species L. acidophilus CH5 and L. casei spp. were observed to present practically double the BSH enzyme activity of the L. acidophilus NCFM. Probiotic supplementation fed the animals did not alter (P > 0.05) food consumption, food efficiency coefficient, weight gain, liver weight index, spleen weight index, atherogenic index, and total cholesterol concentration, HDL-C, VLDL+LDL-C, bile acids and triacylglycerol among the experimental groups. However, the hyperlipidic diet fed the animals did not increase blood cholesterol concentration (P>0.05). The evaluation of the effect of feeding different concentrations of L. casei spp. (104, 106, 10 8 UFC/mL) to xxv normal and hypercholesterolemic rats for 14 days, showed that weight gain (P >0.05), food efficiency coefficient (P<0.01) and blood parameters atherogenic index and serum levels of total cholesterol, triacylglycerol, HDL-C, LDL+VLDL-C) (P<0.01) of the animals fed the hyperlipidic diet were higher than those of the animals fed a basal diet. Regardless of the type of diet, different concentrations of L. casei spp. fed to the animals did not have an effect on these parameters (P>0.05), despite the increase (P<0.01) in liver weight, liver cholesterol concentration, and cholesterol and fecal bile acid concentrations in the animals fed the hyperlipidic diet, compared to those fed the basal diet. It was observed that the dose effect was significant for the liver cholesterol (P<0.01) and fecal cholesterol (P<0.05), regardless of type of diet. However, no significant difference was found (P>0.05) among the different concentrations of L. casei spp. The repetition of the previous experiment using normal and hypercholesterolemic rabbits, but at concentrations105, 107, 10 9 UFC/mL, showed that the blood parameters (P<0.01), liver weight (P<0.01) and liver cholesterol concentration and fecal cholesterol (P<0.01) of the animals fed the hyperlipidic diet were higher than those of the animals fed the basal diet. However, contrary to the rats, the dose effect was not significant (P>0.05) for all the characteristics analyzed. Regarding the dose effect on other functional properties of the alimentary canal in normocholesterolemic rats and rabbits, it was confirmed that the different probiotic concentrations tested increased the Lactobacillus levels (P>0.05) and decreased E. coli levels (P<0.05) and the enzyme β-glucuronidase activity (P<0.01) in rats but not in rabbits (P>0.05). On the other hand, no alteration was found either in spleen weight nor in spleen bacterial counting (P>0.05) but there was an increase in the length of the intestinal villi (P>0.01) in both animal models. Therefore, it was concluded that, although no alteration was verified in the animals’ cholesterolemia, supplemmenting increasing L. casei spp. doses had some effect on cholesterol metabolism, intestinal morphology and intestinal microbiota modulation, increasing the Lactobacillus levels and reducing the E. coli levels for both models studied. / Não foi localizado o cpf do autor.

Acetohydroxyacid synthase is a multisubunit enzyme that catalyses the first committed step in the biosynthesis of the branched chain amino acids viz., valine, leucine and isoleucine. In order to understand the structural basis for the observed allosteric feedback inhibition in AHAS, the regulatory subunit of AHAS isozymes I from E. coli was cloned, expressed, purified and the conditions were optimized for solution NMR spectroscopy. IlvN was found to exist as a dimer both in the presence and absence of the feedback inhibitor. Using high-resolution multidimensional, multinuclear NMR experiments, the structure of the dimeric valine-bound 22 kDa IlvN was determined. The ensemble of twenty low energy structures shows a backbone root mean square deviation of 0.73 ± 0.13 Å and a root mean square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, greater than 98% of the backbone φ, ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain fold that is observed in regulatory domains of metabolic enzymes. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR timescale. Important structural properties of the unliganded state were probed by H-D exchange studies by NMR, alkylation studies by mass spectrometry and other biophysical methods. It was observed that the dynamic unliganded IlvN underwent a coil-to-helix transition upon binding the effector molecule and this inherent conformational flexibility was important for activation and valine-binding. A mechanism for allosteric regulation in the AHAS holoenzyme was proposed. Study of the structural and conformational properties of IlvN enabled a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis. Solution structural studies of 32 kDa Protease-VPg (PVPg) from Sesbania mosaic virus (SeMV) Polyprotein processing is a commonly found mechanism in animal and plant viruses, by which more than one functional protein is produced from the same polypeptide chain. In Sesbania Mosaic Virus (SeMV), two polyproteins are expressed that are catalytically cleaved by a serine protease. The VPg protein that is expressed as a part of the polyprotein is an intrinsically disordered protein (by recombinant expression) that binds to various partners to perform several vital functions. The viral protease (Pro), though possessing the necessary catalytic residues and the substrate binding pocket is unable to catalyse the cleavage reactions without the VPg domain fused at the C-terminus. In order to determine the structural basis for the aforementioned activation of protease by VPg I undertook the structural studies of the 32 kDa PVPg domains of SeMV by solution NMR spectroscopy. NMR studies on this protein were a challenge due to the large size and spectral overlap. Using a combination of methods such as deuteration, TROSY-enhanced NMR experiments and selective ‘reverse-labelling’, the sequence specific assignments were completed for ~80% of the backbone and 13C nuclei. NMR studies on mutants such as the C-terminal deletion mutant, I/L/V to A mutants in VPg domain were conducted in order to identify the residues important for aliphatic-aromatic interactions observed in PVPg. Attempts were made to obtain NOE restraints between Pro and VPg domains through ILV labelled samples; however these proved unsuccessful. It was observed that ‘natively unfolded’ VPg possessed both secondary and tertiary structure in PVPg. However, 30 residues at the C-terminus were found to be flexible. Even though atomic-resolution structure could not be determined, the region of interaction between the domains was determined by comparing NMR spectra of Pro and PVPg. The conditions for reconstitution of the Protease-VPg complex by recombinantly expressed Pro and VPg proteins were standardised. These studies lay the foundation for future structural investigations into the Protease-VPg complex.