The human gut is home to a significant number of microorganisms including the dominant symbiont Bacteroides thetaiotaomicron. This microbe is predicted to possess an array of glycoside hydrolases, majority of which are involved in starch utilization. Presented here is a comparative functional analysis of two alpha-glucosidases, Family 31 Glycoside Hydrolases from Bacteroides thetaiotaomicron. Enzymatic kinetics revealed these enzymes both preferentially cleave alpha-(1,6) linkage in comparison to the predicted alpha-(1,4) and favour maltose derived substrates of longer length.

The human gut is home to a significant number of microorganisms including the dominant symbiont Bacteroides thetaiotaomicron. This microbe is predicted to possess an array of glycoside hydrolases, majority of which are involved in starch utilization. Presented here is a comparative functional analysis of two alpha-glucosidases, Family 31 Glycoside Hydrolases from Bacteroides thetaiotaomicron. Enzymatic kinetics revealed these enzymes both preferentially cleave alpha-(1,6) linkage in comparison to the predicted alpha-(1,4) and favour maltose derived substrates of longer length.

Class of 2017 Abstract / Objectives: Evaluate thymoquinone (TQ) and thymol (THY) inhibitory activities against α-glucosidase enzyme by using an in vitro assay and determine the IC50 (concentration of TQ/THY to inhibit 50% of maximum enzyme activity). Methods: Various concentrations of thymoquinone and thymol were incubated, separately, with one concentration of the substrate - p-nitrophenol-α-D-glucopyranoside (PNPG) (<<Km) in presence of α-glucosidase enzyme. A positive and a negative control consisting of acarbose, and buffer, respectively, were included in the incubation as well. The incubation time was set at 30 min in a 37 °C controlled water bath. The enzyme activity was determined by detecting and quantitating the levels of p-nitrophenol using a spectrophotometer set at 405 nm. The percent inhibition exhibited by any studied drug was calculated as shown in equation 1. % inhibition = Absorbance Substrate Alone – Absorbance of Substrate + Inhibitor Absorbance Substrate Alone Results: Results of the in vitro incubation of thymoquinone, thymol and acarbose revealed “statistically” significant inhibition of -glucosidase (p<0.05). At 400 g/ml, thymoquinone inhibited the enzyme activity by ~52 % whereas the enzyme inhibition by thymol and acarbose were calculated to be ~84% and 57%, respectively. IC50 were tentatively determined although the maxima inhibitions of the inhibitors were not reached fully. IC50s were calculated as 234 μg/ml, 304 μg/ml and 157 μg/ml for each of thymoquinone, thymol and acarbose, respectively. Conclusions: Thymoquinone and thymol do exhibit antagonistic pharmacological activity against α-glucosidase.

La levure Saccharomyces cerevisiae est capable d’utiliser une grande variété de sucres comme source de carbone et d’énergie. La plupart des enzymes impliquées dans l’utilisation de ces sucres sont codées par des gènes issus de familles multigéniques. C’est le cas de la famille IMA identifiée comme impliquée dans l’utilisation de l’isomaltose. Cette famille comprend cinq gènes qui codent pour quatre isomaltases partageant une forte identité de séquence (de 65% à 100 %). Dans ce travail , la diversitéfonctionnelle de la famille IMA a été étudiée, en caractérisant de façon exhaustive in vitro leurs propriétés biochimiques et enzymologiques. Ima1p et Ima2p possèdent des propriétés biochimiques identiques (pH, température, et thermostabilité) mais Ima3p se distingue par rapport à ces deux protéines bien que n’ayant que trois acides de différence avec Ima2p (thermostabilité plus faible). Ima5p quant à elle, est la protéine la plus dissemblable (température optimale plus faible et demi-vie basse dès 37°C). Les quatre isomaltases sont cependant très sensibles au Tris et aux ions Fe3+. Les quatre isoenzymes présentent une préférence pour les disaccharides liés en α-1,6 (isomaltose et palatinose), avec une cinétique de type Michaëlis-Menten et une inhibition par le substrat à une concentration élevée. Les isomaltases Imap sont cependant aussi capables d'hydrolyser les disaccharides α-1,2, α-1,3 et α-1,5 ainsi que les trisaccharides portant une liaison α-1,6, ce qui met en évidence leur ambiguïté de substrat .Nos résultats ont toutefois montré de nombreuses singularités dans cette famille de protéines. Alors que Ima1p et Ima2p présentent des propriétés très semblables, l’activité catalytique de Ima3p est globalement très faible malgré sa forte ressemblance avec Ima2p. Le variant Ima3p_R279Q retrouve des niveaux d'activité proches de ceux d’Ima2p, tandis que la substitution d’une leucine par une proline à la position 240 a permis d’augmenter de manière significative la stabilité d’Ima3p confirmant le rôle des prolines dans la thermostabilité des protéines. L’hydrolyse de l’isomaltose par Ima5p réfute lesconclusions précédemment publiées sur l'exigence d'acides aminés spécifiques pour déterminer la spécificité de α-1,6 puisque le variant IMA5-MQH ne permet pas de restaurer une activité semblable à Ima1p malgré la présence des trois résidus MQH. Nous avons également trouvé qu’Ima5p est inhibé par le maltose suivant une inhibition mixte tandis qu’Ima1p est inhibée de façon compétitive à faible concentration et de manière incompétitive à forte concentration en isomaltose / Most enzymatic systems for sugar uptake and assimilation rely on multigene families in theyeast Saccharomyces cerevisiae. The IMA / MAL family has been used as a model system to study themolecular mechanisms that govern evolution of duplicated genes. The five IMA multigene familymembers encode four isomaltases sharing high sequence identity from 65% to 99%, of which IMA3and IMA4 are 100% identical to encode the same isomaltase. In this work, the functional diversity ofIMA family was further explored, with exhaustive in-vitro characterization of their biochemical andenzymological properties.Ima1p and Ima2p were similar to biochemical properties; Ima3p showed some differences fromthe two proteins; amongst them, Ima5p was the most distant protein. The four isomaltases were highlysensitive to Tris and Fe3+, but were unaffected by the addition or the removal of Ca2+ despiteconservation of the calcium binding site. Besides, four isoenzymes exhibited a preference for the α-(1,6)disaccharides isomaltose and palatinose, with Michaelis-Menten kinetics and inhibition at highsubstrates concentration. They were also able to hydrolyse trisaccharides bearing an α-(1,6) linkage,but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrateambiguity. While Ima1p and Ima2p presented almost identical characteristics, the results neverthelessshowed many singularities within this protein family. In particular, Ima3p presented lower activitiesthan Ima2p despite only 3 different amino acids between these two isoforms. The Ima3p_R279Qvariant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240significantly increased the stability of Ima3p and supported the role of prolines inthermostability.Ima5p presented the lower optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specificamino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibitionby maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitiveat very low isomaltose concentrations and uncompetitive as the substrate concentration increased.The presented Ph.D’s work provided preliminary insights into determining structural factorswithin this family, exemplifying for example the role of proline residues for thermosability. Moreover,it was illustrated that a gene family encoding proteins with strong sequence similarities can lead toenzyme with notable differences in biochemical and enzymological properties.

Masters of Science / Diabetes mellitus (DM) is well known as a group of systemic metabolic disorders with a considerable mortality rate around the world. Hyperglycemia is the main consequence of DM, which results from the shortage in insulin production or degradation of produced insulin. Other internal and external factors including obesity, oxidative stress, and sedentary lifestyle have been also suggested as the causes of DM. Among the well-known existing types of DM, type 1 and 2 are the most common. Treatment of type 1 necessitates insulin injection, while type II can be controlled by physical exercises, diet control in addition to different synthetic antidiabetic drugs. However, their effectiveness is restricted because of the high cost and unfriendly side effects. There is a significant need for producing alternative and more bioactive antidiabetic drugs from natural sources. Natural products are a well-known source for the discovery of new scaffold for drugs discovery, and South Africa is one of the most important megaflora with a high percentage of endemism. South Africa is home to about 245 species of Helichrysum species, which are generally rich sources of secondary metabolites, especially flavonoids, which give them a great value for global acceptability and wide application in the pharmaceutical fields as antibacterial, anti-inflammatory, antioxidant, and antidiabetic agents. The phytochemical investigation of the methanolic extract of H. cymosum resulted in the isolation and identification of seven pure compounds (1-7). Structural characterization of these isolated compounds was conducted using 1D NMR, in comparison with reported spectroscopic data. The in vitro bio-evaluation of H. cymosum against alpha-glucosidase shown that 5 exhibited the highest alpha-glucosidase inhibitory activity with IC50 value of 13 μM, followed by 7 and 3 with IC50 values of 18.16 μM and 44.4 μM respectively. Additionally, strong total antioxidant capacities were displayed by 6 and 2 as ORAC (122.86 ± 0.7 and 91.70 ± 0.4 μM TE/mL) respectively as well as 5 and 7 as FRAP (1006.34 ± 1.7; 977.79 ± 0.8) μM AAE/g. This is the first scientific report to be carried out on alpha-glucosidase inhibitory activities and antioxidant capacities of H. cymosum constituents. The findings suggest that these compounds might become prominent natural candidates to inhibit alpha-glucosidase as well as oxidative stress related to diabetes with the prospect to be employed in the formulation of diabetes drugs upon further biological studies.

Introdução: Polifenóis não-extraíveis (NEPPs) são uma fração de polifenóis que não são extraídos da forma convencional por estarem associados à parede celular de produtos de origem vegetal. Um corpo crescente de estudos tem evidenciado seus potenciais efeitos benéficos, especialmente associados à saúde intestinal e interações com a microbiota. O guaraná (Paullinia cupana), fruto típico da biota amazônica, é conhecidamente rico em polifenóis da família dos flavanóis, mas ainda existe uma lacuna a respeito da fração de polifenóis não-extraíveis em sua composição. Objetivo: Caracterizar a fração de polifenóis não-extraíveis quanto a sua composição química, e avaliar sua potencial capacidade de inibição enzimática. Métodos: O guaraná em pó foi submetido a extração aquo-orgânica para obtenção da fração extraível, e o resíduo proveniente dessa extração foi submetido a hidrólise ácida e hidrólise básica para obtenção dos NEPPs. A capacidade redutora total (CRT) foi quantificada pelo método de Folin-Ciocalteu. A quantificação de taninos condensados foi realizada pelo método de Porter. A determinação do perfil de fenólicos foi realizada por HPLC-ECD e LC-MS para as frações extraíveis e hidrolisáveis, e MALDi-TOF/TOF para a fração condensada. Os testes enzimáticos foram realizados com base na cinética de estado estacionário. Os testes estatísticos foram realizados utilizando softwares Excel e SPSS. Resultados: O perfil de fenólicos para a fração extraível consiste na presença de catequina e epicatequina como componentes majoritários, com 5,45 ± 0,15 e 5,95 ± 0,22 mg/g de guaraná em pó (base seca), respectivamente, além de proantocianidinas B1 e B2 e trímero de tipo A. Já o perfil fenólico da fração não-extraível contém uma mistura complexa de monômeros como catequina, leucoantocianidina, cianidina e delfinidina. A fração NEPP também contém dímeros, trímeros, tetrâmeros e pentâmeros de flavanóis, tanto de tipo A quanto de tipo B, com alta variabilidade de grau de hidroxilação. O ensaio enzimático com &alpha;-glicosidase resultou em valores de IC50 de 9,504 e 1,624 &micro;g EAG/mL para a fração extraível e a não-extraível, respectivamente. O modo de inibição para ambas as frações foi classificado como misto, com valores de Ki e K\'i de 0,403 e 1,735 &micro;g/mL para a fração extraível e 0,287 e 0,847 &micro;g/mL para a fração não-extraível. Conclusões: A fração de polifenóis não-extraíveis possui composição variada e complexa quando comparada a fração extraível, e possui potencial de inibição de &alpha;-glicosidase que deve ser explorado de maneira mais aprofundada, uma vez que tal potencial é de interesse para o controle de doenças crônicas como o diabetes tipo 2. / Introduction: Non-extractable polyphenols (NEPPs) are a portion of polyphenols that cannot be extracted in the conventional way due to being associated with the cell wall of products of plant origin. A growing number of studies have been showing its potential beneficial effects, especially in relation to gut health and microbiota interactions. The guarana (Paullinia cupana), a fruit native of the Amazon rainforest, is known to be rich in polyphenols from the flavanol family, but there is still a gap about non-extractable polyphenols in its composition. Objective: Characterize the non-extractable polyphenol portion in relation to its chemical composition and evaluate its enzymatic inhibition capacity. Methods: The extractable fraction was obtained by aqueous-organic extraction, and the residue from this extraction was treated with acid and alkaline hydrolysis to obtain the NEPPs. The total reducing capacity (TRC) was quantified by the Folin-Ciocalteu method. The quantification of condensed tannins was performed with the Porter method. The phenolic profile was determined by HPLC-ECD and LC-MS for the extractable and hydrolysable fractions, and MALDi-TOF/TOF for the condensed fraction. The enzymatic assay was carried out using steady-state kinetics. The statistical tests were performed using Excel and SPSS. Results: The phenolic profile of the extractable fraction consists of catechin and epicatechin as major components with 5,45 ± 0,15 and 5,95 ± 0,22 mg/g guarana powder (dry weight), respectively, besides B1 and B2 proanthocyanidins and type A trimer. The phenolic profile of the non-extractable fraction contains a complex mixture of monomers like catechin, leucoanthocyanidin, cyanidin, and delphinidin. The NEPP fraction also contains type A and type B dimers, trimers, tetramers, and pentamers of flavanols, with high variability of the degree of hydroxylation. The &alpha;-glucosidase enzymatic assay had IC50 values of 9,504 and 1,624 Introduction: Non-extractable polyphenols (NEPPs) are a portion of polyphenols that cannot be extracted in the conventional way due to being associated with the cell wall of products of plant origin. A growing number of studies have been showing its potential beneficial effects, especially in relation to gut health and microbiota interactions. The guarana (Paullinia cupana), a fruit native of the Amazon rainforest, is known to be rich in polyphenols from the flavanol family, but there is still a gap about non-extractable polyphenols in its composition. Objective: Characterize the non-extractable polyphenol portion in relation to its chemical composition and evaluate its enzymatic inhibition capacity. Methods: The extractable fraction was obtained by aqueous-organic extraction, and the residue from this extraction was treated with acid and alkaline hydrolysis to obtain the NEPPs. The total reducing capacity (TRC) was quantified by the Folin-Ciocalteu method. The quantification of condensed tannins was performed with the Porter method. The phenolic profile was determined by HPLC-ECD and LC-MS for the extractable and hydrolysable fractions, and MALDi-TOF/TOF for the condensed fraction. The enzymatic assay was carried out using steady-state kinetics. The statistical tests were performed using Excel and SPSS. Results: The phenolic profile of the extractable fraction consists of catechin and epicatechin as major components with 5,45 ± 0,15 and 5,95 ± 0,22 mg/g guarana powder (dry weight), respectively, besides B1 and B2 proanthocyanidins and type A trimer. The phenolic profile of the non-extractable fraction contains a complex mixture of monomers like catechin, leucoanthocyanidin, cyanidin, and delphinidin. The NEPP fraction also contains type A and type B dimers, trimers, tetramers, and pentamers of flavanols, with high variability of the degree of hydroxylation. The &alpha;-glucosidase enzymatic assay had IC50 values of 9,504 and 1,624 Introduction: Non-extractable polyphenols (NEPPs) are a portion of polyphenols that cannot be extracted in the conventional way due to being associated with the cell wall of products of plant origin. A growing number of studies have been showing its potential beneficial effects, especially in relation to gut health and microbiota interactions. The guarana (Paullinia cupana), a fruit native of the Amazon rainforest, is known to be rich in polyphenols from the flavanol family, but there is still a gap about non-extractable polyphenols in its composition. Objective: Characterize the non-extractable polyphenol portion in relation to its chemical composition and evaluate its enzymatic inhibition capacity. Methods: The extractable fraction was obtained by aqueous-organic extraction, and the residue from this extraction was treated with acid and alkaline hydrolysis to obtain the NEPPs. The total reducing capacity (TRC) was quantified by the Folin-Ciocalteu method. The quantification of condensed tannins was performed with the Porter method. The phenolic profile was determined by HPLC-ECD and LC-MS for the extractable and hydrolysable fractions, and MALDi-TOF/TOF for the condensed fraction. The enzymatic assay was carried out using steady-state kinetics. The statistical tests were performed using Excel and SPSS. Results: The phenolic profile of the extractable fraction consists of catechin and epicatechin as major components with 5,45 ± 0,15 and 5,95 ± 0,22 mg/g guarana powder (dry weight), respectively, besides B1 and B2 proanthocyanidins and type A trimer. The phenolic profile of the non-extractable fraction contains a complex mixture of monomers like catechin, leucoanthocyanidin, cyanidin, and delphinidin. The NEPP fraction also contains type A and type B dimers, trimers, tetramers, and pentamers of flavanols, with high variability of the degree of hydroxylation. The &alpha;-glucosidase enzymatic assay had IC50 values of 9,504 and 1,624 &micro;g GAE/mL for the extractable and non-extractable fraction, respectively. The mode of inhibition was classified as mixed for both fractions, with Ki and K\'i values of 0,403 and 1,735 &micro;g/mL for the extractable fraction and 0,287 and 0,847 &micro;g/mL for the non-extractable fraction. Conclusions: The non-extractable polyphenols fraction has a varied and complex composition when compared to the extractable fraction, and it has a &alpha;-glucosidase inhibition potential that must be explored in a more detailed fashion since said potential is of interest for the control of chronic diseases such as type 2 diabetes. g GAE/mL for the extractable and non-extractable fraction, respectively. The mode of inhibition was classified as mixed for both fractions, with Ki and K\'i values of 0,403 and 1,735 &micro;g/mL for the extractable fraction and 0,287 and 0,847 &micro;g/mL for the non-extractable fraction. Conclusions: The non-extractable polyphenols fraction has a varied and complex composition when compared to the extractable fraction, and it has a &alpha;-glucosidase inhibition potential that must be explored in a more detailed fashion since said potential is of interest for the control of chronic diseases such as type 2 diabetes. g GAE/mL for the extractable and non-extractable fraction, respectively. The mode of inhibition was classified as mixed for both fractions, with Ki and K\'i values of 0,403 and 1,735 &micro;g/mL for the extractable fraction and 0,287 and 0,847 &micro;g/mL for the non-extractable fraction. Conclusions: The non-extractable polyphenols fraction has a varied and complex composition when compared to the extractable fraction, and it has a &alpha;-glucosidase inhibition potential that must be explored in a more detailed fashion since said potential is of interest for the control of chronic diseases such as type 2 diabetes.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Diabetes mellitus (DM) is a group of non-infectious diseases that cause hyperglycemia. DM symptoms were first clinically described by ancient Greek physicians whose prescriptions included plant-based remedies. Today, DM affects >400 million people globally and prevalence rates are rapidly increasing in developing countries where basic healthcare relies on local knowledge of botanical remedies. Many developing countries are home to diverse peoples and plants—providing fodder for varied plant-selection strategies and unique botanical pharmacopoeias. I addressed the plant-selection strategies used in a multi-ethnic, developing country, Trinidad and Tobago (T&T), to ascertain their role in shaping the local antidiabetic pharmacopoeia and to assess their benefits and risks in identifying safe and useful remedies. Using literature reviews, field surveys, and laboratory bioassays, I completed three categories of analysis. Ethnobotanical analyses showed that T&T’s antidiabetic pharmacopoeia is primarily of recent origin as >50% of the 48 historical DM remedies were Neotropical natives, including congenerics of well-known medicinal Paleotropical genera. Nevertheless, conservative knowledge transmission was also evident as several Paleotropical species of T&T’s pharmacopoeia, including Momordica charantia and Catharanthus roseus were also used in Africa, India and across the Caribbean. Paleotropical natives with a long history of use are likely to be safer remedies. Ethno-medicinal analyses of the pre- and post-2000 DM remedies of T&T, totaling 99 species, suggest that the centuries-old hot/cold folk disease-model was the model predominantly used in plant-selection. Parallels found between T&T folk concepts and biomedical mechanisms of DM provide probable bases for efficacy but the chronic use of purgatives and bitter-tasting plants is likely to be risky. Phytochemical analyses revealed that 69% of the tested plant extracts contained phenolic compounds, with more than half producing >80% alpha-glucosidase inhibition. Phenolic content and alpha-glucosidase inhibition were strongly correlated among food plants used as medicines, suggesting higher probability of selection as a result of non-target effects. The medicinal use of food plants may provide the best margins of safety and efficacy in identifying antidiabetic remedies. Together, these analyses showed how culture-specific plant-selection strategies can identify safe, useful remedies for developing countries to address their increasing DM prevalence in a cost-effective and sustainable manner.