Dissertations / Theses on the topic 'Methionase enzyme'

Human methionine synthase reductase (MSR) is a 78-kDa diflavin enzyme involved in folate and methionine metabolism. It regenerates the cofactor of methionine synthase (MS), cob(II), to reduce inactive MS. MSR and one of its the FAD/NADPH binding domain were cloned as GST-tagged fusion proteins for expression and purification in Escherichia coli. And a 1.9 Å Crystals of the FAD/NADPH binding domain of MSR with and without NADP+ were produced and carried out X-ray diffraction experiment and the structure of the crystal was solved by molecule replacement method. The activation domain of human MS was also expressed and purified in Escherichia coli and crystallization conditions determined. A new expression vector for full-length MSR, which contains a N-terminal GST tag, and C-terminal 6× His tag, was constructed and validated by sequencing, restriction enzymes digestion and successfully expressed in E. coli and Yeast Pichia pastoris. Based on the structure information, site-directed mutagenesis on the two positions Asp652 and trpytophan697 of MSR were designed and completed. The variants D652A, D652R, D652N of the FAD/NADPH binding domain of MSR and the variants D652A,D652R,D652N, W696A,W697H of the full-length MSR were cloned and expressed in BL21 (E. coli). The proteins of these mutants were purified by affinity chromatography, anion exchange chromatography and gel filtration chromatography. And the kinetic studies on these variants of MSR were investigated in steady state kinetic study, steady state inhibition studies, stopped-flow pre steady-state kinetic and redox potential studies. Compared with the data of the wild type MSR, the turnover number of variants all decreased, the catalytic ability become lower and the midpoint potential of cofactor FAD occurred positive shift. Both 2'5-ADP and NADP+ were competitive inhibitors for variants of MSR. However, 2'5'-ADP was relative strong inhibitor than NADP+. All the data on variants of MSR suggested the Asp652 and tryptophan697 were two important structural and function determinant of MSR. To investigate the dynamic properties of EPR, ENDOR and ESMME are used to investigate the existence of the semiquinone flavin cofactors, FAD and FMN, and the hyperfine coupling arising from the interaction of some nuclei with the unpaired electron spin. ELDOR spectroscopy was applied to measure the distance between the FAD and FMN in MSR under the binding of 2', 5'-ADP, NADP and the activation domain of MS to further check the conformational change of MSR.

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while similar to 3.6 x 10(3) CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia colt TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized Glade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols. The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro were advanced. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and, potentially, in vivo applications. In order to further address the instability of AdoMet and analogs thereof, MAT-catalyzed reactions utilizing Met and ATP isosteres generated highly stable AdoMet isosteres that were capable of downstream utilization by MTs. Finally, the development, use, and results of a high-throughput screen identified mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.

Radical SAM enzymes are ancient, essential enzymes. They perform radical chemical reactions in virtually all living organisms and are involved in producing antibiotics, generating greenhouse gases, human health, and likely many other essential roles that have yet to be established. A wide variety of reactions have been characterized from this group of enzymes, including hydrogen abstractions, the transferring of methylthio groups, complex cyclization and rearrangement reactions, and others. However, many radical SAM enzymes have yet to be identified or characterized. There have been great leaps forward in the amount of enzyme sequences that are available in public databases, but experiments to investigate what chemical reactions the enzymes perform take a great deal of time. In our work, we utilize Hidden Markov Models to identify possible radical SAM enzymes and predict their possible functions through BLAST alignments and homology modelling. We also explore their distribution across the tree of life and determine how it is correlated with organism oxygen tolerances, because the core iron-sulfur cluster is oxygen sensitive. Trends in the abundances of radical SAM enzymes depending on oxygen tolerances were more apparent in prokaryotes than in eukaryotes. Although eukaryotes tend to have fewer radical SAM enzymes than prokaryotes, we were able to analyze uncharacterized radical SAM enzymes from both an aerobic eukaryote (Entamoeba histolytica) and a eukaryote capable of oxygenic photosynthesis (Gossypium barbadense), and predict the reactions they catalyze. This work sets the stage for the functional characterization of these essential yet elusive enzymes in future laboratory experiments.
Master of Science in Life Sciences
Radical SAM enzymes are ancient, essential enzymes that perform chemical reactions in virtually all living organisms. We do know that they are involved in producing antibiotics, human health, and generating greenhouse gases. We also know that there are many radical SAM enzymes whose functions remain a mystery. There have been great leaps forward in the amount of enzyme sequences that are available in public databases, but experiments to investigate what chemical reactions enzymes perform take a great deal of time. The experiments are especially difficult for radical SAM enzymes because the oxygen we breathe can break the enzymes down in a laboratory. In our work, we utilize computational techniques to identify possible radical SAM enzymes and predict what reactions they might catalyze. Because these enzymes are vulnerable to oxygen in laboratory environments, we also explore whether organisms that breathe oxygen have fewer of these enzymes than organisms that perform anaerobic respiration instead. We found that does seem to be the case in microbes like bacteria and archaea, but the results were not as consistent for eukaryotes. We then chose radical SAM enzymes we had identified from both an aerobic eukaryote (Entamoeba histolytica) and a eukaryote capable of producing oxygen (Gossypium barbadense), and predicted the reactions they catalyze. This work sets the stage for the functional characterization of these essential yet elusive enzymes in future laboratory experiments.

Thesis (M.S.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on May 12, 2009) Includes bibliographical references.

Wyosine derivatives are highly complex modified ribonucleic acid (RNA) bases found in archaea and eukarya. They are a modification of a genetically encoded guanosine found at position 37 of phenylalanine encoding transfer ribonucleic acid (tRNA). The second step in the biosynthesis of all wyosine derivatives, in both archaea and eukarya, is the transformation of N-methylguanosine to 4-demethylwyosine by the radical S-adenosyl-l-methionine enzyme TYW1. When these studies were initiated, the substrate of TYW1 was unknown. Four possible substrates; acetyl CoA, acetyl phosphate, phosphoenolpyruvate, and pyruvate; were tested for activity. Only incubation with pyruvate led to production of 4-demethylwyosine. As only two new carbons are incorporated into the RNA base at this step, ¹³C isotopologues were used to identify the carbons that are transferred into 4-demethylwyosine. These experiments revealed that C2 and C3 of pyruvate are incorporated into 4-demethylwyosine, with C1 lost as an unknown byproduct. Utilizing pyruvate containing deuteriums in place of protons on the C3 carbon, the regiochemistry of the addition was determined. It was found that C3 forms the methyl group of 4-demethylwyosine and C2 becomes the bridging carbon in the imidazoline ring. The site of hydrogen atom abstraction by 5'-deoxyadenosyl radical was identified as the N-methylguanosine methyl group through the use of tRNA containing a deuterated methyl group. The putative mechanism for this transformation involved the formation of an enzyme substrate Schiff base through a conserved lysine residue. Utilizing sodium cyanoborohydride a Schiff base was trapped between TYW1 and pyruvate. The mass of the trapped adduct responded as expected when different isotopologues of pyruvate were used, demonstrating that it is due to pyruvate. Moreover, the fragment of TYW1 that contained the trapped adduct contained two lysine residues, one of which was shown to be required for activity both in vivo and in vitro. It was initially proposed that TYW1 contained two iron-sulfur clusters, and then subsequently shown to have two 4Fe-4S clusters. Site directed mutagenesis, along with iron and sulfide analysis identified the cysteines; as C26, C39, and C52; coordinating the second 4Fe-4S cluster. This study identified pyruvate as the substrate of TYW1, and provided evidence for key steps in the transformation of N-methylguanosine to 4-demethylwyosine.

Thesis (Ph. D.)--University of Sydney, 2009.
Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Pathology, Faculty of Medicine. Title from title screen (viewed Sept. 30, 2009) Includes bibliography. Also available in print form.

Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Members of the S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily catalyze a myriad of diverse and challenging biotransformations using a [4Fe-4S] cluster and a molecule of AdoMet to initiate radical. In this thesis, we used a combination of crystallographic and biochemical methods to identify the use of covalent catalysis and polar reactions in two AdoMet radical enzymes that catalyze the key steps in the biosynthesis of the tRNA modified bases wybutosine and queuosine. TYWI catalyzes the formation of the characteristic imidazopurine ring of wybutosine through a disputed mechanism. Here, we have garnered support for one of the proposed mechanisms, through the identification and characterization of a Schiff base between a catalytically essential lysine residue and the substrate pyruvate. The ability of TYWI to form and possibly use a Schiff base presents the first instance of a covalent catalysis in the mechanism of an AdoMet radical enzyme. In an attempt to obtain a snapshot of the active site of the queuosine biosynthetic enzyme, QueE, with AdoMet and a substrate analog, 6-carboxypterin (6-CP), we uncovered a covalent adduct between AdoMet and 6-CP. Further investigation of the mechanism by which this adduct was formed revealed a polar mechanism instead of a radical one. This result highlights the ability for AdoMet radical enzymes to use the same active site for two different reactions, polar and/or radical reactions. The unifying characteristics of this superfamily include the canonical CX₃CX[phi]C cluster-binding motif and a partial ([beta]/[alpha]X) 6 triose isomerase phosphate (TIM) barrel. Work in this thesis presents the structural characterization of a third QueE ortholog from Escherichia coli. Together, these three QueE orthologs revealed different variations in the core barrel architecture, which may influence binding of the biological reductant Flavodoxin. This variance in the core AdoMet radical fold emphasizes the structural diversity of this superfamily. On the other hand, we see conservation of an overall three-domain architecture for the maturation of ribosomally synthesized and post-translationally modified natural products, underlining the importance of this architecture for catalysis.
by Tsehai A.J. Grell.
Ph. D. in Biological Chemistry

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first protocol tackles this problem by developing a way to obtain active untagged recombinant PRMT proteins. The second protocol describes a fast and efficient method for quantitative measurement of AdoMet-dependent methyltranseferase activity with protein substrates. In addition to being very sensitive, this method decreases the processing time for the analysis of PRMT activity to a few minutes compared to weeks by traditional methods, and generates 3000-fold less radioactive waste. We then used these methods to investigate the effect of truncating the NT of human PRMT1 variant 1 (hPRMT1-V1) on enzyme activity, protein-protein interactions, and substrate specificity. Our studies show that the NT of hPRMT1-V1 influences enzymatic activity and protein-protein interactions. In particular, methylation of a variety of protein substrates was more efficient when the first 10 amino acids of hPRMT1v1 were removed, suggesting an autoinhibitory role for this small section of the N-terminus. Likewise, as portions of the NT were removed, the altered hPRMT1v1 constructs were able to interact with more proteins. Overall, my studies suggest the the sequence and length of the NT of hPRMT1v1 is capable of enforcing specific protein interactions.

Sabe-se que concentrações elevadas de homocisteína (Hcy) estão associadas com o aumento do risco de doenças cardiovasculares e com o dano celular causado pela formação das espécies ativas de oxigênio (EAO). Sabe-se também que o estrogênio atua como antioxidante não enzimático envolvido na proteção cardiovascular. Foram objetivos deste trabalho avaliar o efeito da homocistinúria sobre parâmetros de estresse oxidativo em tecido cardíaco; e avaliar o efeito da hiperhomocisteinemia (HHcy) sobre parâmetros de estresse oxidativo e hemodinâmicos em ratas com e sem estrogênio. Este trabalho foi divido em 4 experimentos. No experimento 1 foram utilizados 16 animais divididos em 2 grupos (n= 8/grupo): controle e Hcy. Estes animais receberam tratamento crônico do 6° dia ao 28° dia de vida com doses crescentes de Hcy e foram mortos 1 hora após a última dose.No experimento 2 foram utilizados 30 ratos, divididos em 4 grupos: Salina (n=8); Folato (n=6); Hcy (n=9) e Folato+Hcy (n=7). Estes animais receberam ácido fólico e/ou Hcy do 6° ao 28º dias de vida e foram mortos aos 80 dias de vida. No experimento 3, foram utilizados 64 animais divididos em 6 grupos (n= 8/grupo): NAIVE; NAIVE+Hcy; Sham; Sham+Hcy; castrada e castrada+Hcy. Estes animais foram castrados no 50° dia de vida e, após uma semana, receberam tratamento agudo com Hcy de 8 em 8 horas por 72 horas e foram mortos 1 hora após a última dose. No experimento 4, foram utilizados 32 animais divididos em 4 grupos (n= 8/grupo): controle, castrado, metionina e castrado+metionina. Estes animais foram castrados no 70° dia de vida, receberam metionina na água de beber por 30 dias e foram mortos logo após o final do tratamento. No modelo de homocistinúria (experimento 1), não foram observadas alterações na lipoperoxidação (LPO) cardíaca nos ratos com 28 dias. No entanto, as atividades das enzimas antioxidantes SOD e GST estavam aumentadas no grupo Hcy. Como este é um tratamento crônico, provavelmente estas enzimas estão aumentadas de forma a minimizar o dano oxidativo causado pela Hcy. Já no experimento 2, avaliou-se o efeito da Hhcy a longo prazo, em animais com 80 dias. Houve aumento na LPO dos animais tratados com Hcy que foi previnida com a administração de folato. A redução da LPO na presença do folato confirma sua capacidade de minimizar o dano causado pela Hcy. Observamos ainda adiminuição na atividade das enzimas GST e catalase nos animais tratados com Hcy o que resultaria em aumento da concentração de peróxido de hidrogênio no tecido cardíaco. O tratamento com folato previniu a atividade das enzimas antioxidantes. A partir dos resultados encontrados, podemos sugerir que os níveis de Hcy podem ser reduzidos com folato, uma vez que altas doses de folato reduziram consideravelmente os níveis de estresse oxidativo gerado pela Hcy. No modelo de HHcy aguda (experimento 3), o estresse oxidativo cardíaco aumentou em função da administração de Hcy no grupo sem estrogênio. Este resultado se correlaciona positivamente com a pressão arterial (PA), ou seja, os animais com maior LPO também apresentaram uma maior PA. Este efeito não foi observado nos grupos com níveis estrogênicos fisiológicos. Acredita-se que estes resultados sejam devidos à proteção antioxidante oferecida pelo estrogênio. Além disso, observamos uma diminuição na atividade da GST nos animais castrados+Hcy, o que pode estar contribuindo para o dano oxidativo observado. Já no modelo de HHcy causado pelo consumo de metionina (experimento 4), observamos um aumento na PDFVE no grupo castrado+metionina que se correlaciona negativamente com os metabólitos do NO. Este resultado mostra que os animais que tiveram disfunção ventricular apresentaram uma menor biodisponibilidade do NO. Neste modelo observamos também um aumento no dano oxidativo cardíaco em função da administração de metionina no grupo sem estrogênio. Este resultado se correlaciona positivamente com a PDFVE, ou seja, os animais com maior LPO também apresentam uma maior pressão ventricular diastólica, indicando uma possível participação do estresse oxidativo na disfunção ventricular. Estes animais ainda apresentaram um aumento na atividade das enzimas antioxidantes GST e GPx no grupo castrado+metionina, sugerindo que o tratamento crônico levou a uma adaptação do sistema antioxidante enzimático na ausência do estrogênio.
It is known that high concentrations of homocysteine (Hcy) are associated with the increase of risk of cardiovascular disease and of cellular damage caused by the formation of reactive oxygen species (ROS). It is also known that the estrogen acts as a non enzymatic antioxidant involved in cardiovascular protection. In this work we evaluated the effect of homocystinuria on myocardial oxidative stress parameters, and the effect of hyperhomocysteinemia (HHcy) on the same parameters and also on hemodynamics in rats with and without estrogen. Four experiments were performed. In experiment number one, sixteen animals were divided in 2 groups (n=8/group): control and Hcy. These animals received chronic treatment from the 6th to the 28th day of life with increasing doses of Hcy and were killed one hour after the last dose. In the second experiment, thirty rats were divided into four groups: Saline (n=8); Folate (n=6); Hcy (n=9) and Folate+Hcy (n=7).These animals had received folic acid and/or Hcy from the 6th to the 28th day of life and had been killed with 80 days of life. In the third experiment, fourty eight animals were divided in 6 groups (n=8/group): NAIVE; NAIVE+Hcy; Sham; Sham+Hcy; castrated and castrated+Hcy. These animals were castrated in the 50th day of life and, after one week, they received an acute treatment with Hcy for 72 hours at each eight hours and were killed one hour after the last dose. In the fourth experiment, thirty two animals were divided into four groups (n=8/group): control, castrated, methionine and castrated+methionine. These animals had been castrated in the 70th day of life, and received methionine in drinking water for 30 days and were killed at the end of treatment. In the homocystinuria model (experiment number one), there were no signs of alterations in lipid peroxidation (LPO) in 28 day rats. However, antioxidant enzyme activities of SOD and GST were increased in Hcy group. As this is a chronic treatment, probably these enzymes are increased to minimize the oxidative damage caused by Hcy. In the second experiment, the effect of the homocystinuria was evaluated in animals with 80 days. It was observed an increase in LPO in the animals that had received Hcy, but it returned to control values with folate administration. The reduction of LPO in the presence of folate confirms its capacity of minimize the damage caused by Hcy. We also observedreduction in the enzyme activities of GST and catalase in the animals receiving Hcy, which also returned to the control values with the administration of folate. It is possible that Hcy increases the hydrogen peroxide concnetration in the myocardium of these animals. From the results obtained, we can suggest that the levels of Hcy can be reduced with folate, since high doses of folate had significantly reduced the levels of oxidative stress caused by Hcy. In the acute model of HHcy (experiment number three), myocardial oxidative stress increased due to the administration of Hcy in the group without estrogen. This result has a positive correlation with mean arterial pressure (MAP), that is, the higher LPO, the higher MAP. This effect was not observed in groups with physiological estrogens levels. It is possible that these findings are related to the antioxidant protection offered by estrogen. Moreover, we observed a reduction in the activity of GST in the group castrated+Hcy, which can be contributing for the oxidative damage observed. In the HHcy model caused by the consumption of methionine (experiment number four), we observedThis result has a negative correlation with the nitric oxide metabolites (Nox) showing that the animals that had an increased ventricular diastolic pressure had presented lesser NO bioavailability. In this model we also observed an increase in the myocardial oxidative stress due to the administration of methionine in the group without estrogen. This result has a positive correlation with LVEDP, that is, the animals with enhanced LPO also presented high ventricular diastolic pressure, indicating a possible participation of oxidative stress in ventricular dysfunction. These animals also presented an increase in the activities of GST and GPx in group castrated+methionine, suggesting that chronic treatment with methionine leads to an adaptation of the enzymatic antioxidant system in the absence of the estrogen.

Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli. RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V. RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity. These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

Chez les plantes, l'atome de soufre de la methionine derive de la cysteine par une serie de reactions dites de transsulfuration faisant intervenir la cystathionine et l'homocysteine comme intermediaires. En presence de cysteine et d'o-phosphohomoserine, la cystathionine gamma-synthase catalyse une synthese de cystathionine qui est ensuite clivee en homocysteine, pyruvate et ammoniac par la cystathionine beta-lyase. La mise au point de techniques de dosage tres sensibles de ces activites enzymatiques a permis de demontrer que la cystathionine gamma-synthase et la cystathionine beta-lyase sont presentes uniquement dans le stroma des chloroplastes. Ces enzymes ont ensuite ete purifiees a l'homogeneite a partir de chloroplastes de feuilles d'epinard et leurs proprietes cinetiques et physicochimiques ont ete definies. La cystathionine gamma-synthase et la cystathionine beta-lyase jouent un role primordial dans le metabolisme cellulaire puisque l'inhibition de l'une ou l'autre de ces enzymes par la propargylglycine ou l'aminoethoxyvinylglycine est letale pour la plante. Un adnc codant pour la cystathionine beta-lyase d'arabidopsis thaliana a ensuite ete clone par complementation fonctionnelle d'un mutant d'e. Coli deficient en activite enzymatique endogene. Cette proteine est synthetisee sous la forme d'un precurseur polypeptidique de 50,4 kda qui est importe et clive dans le chloroplaste pour obtenir une sous-unite mature de 46 kda. La cystathionine beta-lyase d'a. Thaliana a finalement ete surproduite dans e. Coli. La purification de la proteine recombinante en grande quantite permet d'initier une etude structurale de l'enzyme ainsi que le developpement d'un test de criblage automatise de molecules chimiques en vue de la mise au point d'herbicides nouveaux et specifiques

Les Méthionine sulfoxyde réductases (Msr) catalysent la réduction spécifique des méthionine sulfoxydes (Met-O) en méthionines (Met). Elles sont impliquées dans la résistance des cellules à un stress oxydant et dans la virulence des bactéries pathogènes du genre Neisseria. Cette famille d'enzyme se compose de trois classes, les MsrA et B, structuralement distinctes, et présentant une stéréosléctivité respectivement pour l'isomère S et R de la fonction sulfoxyde du substrat. Une troisième classe, découverte récemment, et appelée fRMsr, catalyse la réduction spécifique de la forme libre de l'isomère R de la fonction sulfoxyde. La fRMsr appartient à la famille des domaines GAF, généralement impliqués dans la signalisation cellulaire, et les fRMsr représentent le premier domaine GAF présentant une activité enzymatique. Les études réalisées au cours de ma thèse sur la fRMsr de Neisseria meningitidis ont permis de montrer que : 1) fRMsr de N. meningitidis présente un mécanisme catalytique identique à MsrA/B avec la formation d'au moins un pont disulfure intramoléculaire Cys84-Cys118 réduit par la thiorédoxine (Trx) ; 2) La Cys118 est le résidu catalytique sur lequel l'intermédiaire acide sulfénique doit se former ; 3) L'étape réductase est l'étape cinétiquement déterminante du mécanisme à deux étapes conduisant à la formation du pont disulfure Cys84-Cys118. La combinaison de l'analyse des résultats cinétiques, et de la structure tridimensionnelle de la fRMsr de N. meningitidis en complexe avec le substrat ont permis de montrer : 1) L'existence d'un site de reconnaissance oxyanion impliqué dans la stabilisation de la fonction carboxylate ; 2) Un rôle de la fonction carboxylate du résidu Asp143 dans la catalyse de l'étape réductase ; 3) Le résidu Glu125 est impliqué dans la reconnaissance et/ou le positionnement du substrat Met-O probablement via la stabilisation du groupement NH3+ ; 4) Un rôle du résidu Asp141 dans le positionnement des résidus Asp143 et Glu125 ; 5) le noyau indole du Trp62 est impliqué dans la stabilisation du groupe méthyle-[epsilon]
Methionine sulfoxide reductases (Msr) catalyze the specific reduction of methionine sulfoxides (Met-O) into methionine (Met). They are involved in cell defences against oxidative stress and virulence of pathogenic bacteria of Neisseria genius. This family of enzymes consists of three classes, MsrA and MsrB, structurally-unrelated, Specific for the S and the R epimer of the sulfoxide function of the substrate, respectively. A third class, recently discovered and called fRMsr, selectively reduce the free form of the R epimer of the sulfoxide function. The fRMsr belongs to the family of GAF domains, they are usually involved in cell signaling, and fRMsr represent the first GAF domain to show enzymatic activity. The studies of the Neisseria meningitidis fRMsr have shown that: 1) The Neisseria meningitidis fRMsr have a identical catalytic mechanism to MsrA and MsrB with the formation of at least one intramolecular disulfide bond, Cys84-Cys118 reduced by thioredoxin (Trx) ; 2) The Cys118 is demonstrated to be the catalytic Cys on which a sulfenic acid is formed ; 3) The Reductase step is the rate determining step of the mechanism leading to the formation of the disulfide bond Cys84-Cys118. The combination of the biochemical and kinetics data, and the examination of the 3D structure of the N. meningitidis fRMsr in complex with its substrate shown: 1) an oxyanion hole involved in the accommodation of the carboxylate group ; 2) the carboxylate group of the Asp143 residue involved in the catalysis of step reductase, and 3) The Glu125 residue involved in the recognition and/or positioning of the Met-O probably by the stabilization of the NH3+; 4) the Asp141 residue involved in the positioning of Asp143 and Glu125 residues ; 5) the indole ring of the Trp62 residue involved in stabilizing of the epsilon-methyl group

Sous l'effet de glycopeptides extraits de parois du mycelium de (phytophtora parantica nicotianae) et appeles eliciteurs, les cellules de tabac en culture synthetisent de grandes quantites d'ethylene. L'etude de l'hormone aux differentes etapes de sa voie de biosynthese montre une stimulation rapide et transitoire de l'activite acc-synthase, vraisemblablement par synthese de novo de l'enzyme, entrainant l'augmentation du taux intracellulaire d'acc sans pour autant modifier celui du n-malonyl-acc. (. . . )

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2016
Malaria is a disease caused by protozoan parasites of the genus Plasmodium that are transmitted to humans by infected female Anopheles mosquitoes. Despite countless efforts toward eradication malaria still remains one of the most prevalent infectious diseases, constituting a major public health concern. The available antimalarial drugs are insufficient to control and eradicate malaria, mostly due to the emergence of drug-resistant parasites. Thus, the development of novel intervention strategies is critical to achieve eradication. As an obligatory intracellular pathogen, Plasmodium establishes close interactions with its host that are crucial to ensure parasite development and survival, one of such is the methionine metabolism. Methionine is an essential amino acid and, as for most living organisms, Plasmodium lacks the ability to synthesize methionine de novo. During the blood-stage of infection Plasmodium obtains methionine mainly through haemoglobin digestion. However, how Plasmodium obtains methionine during the liver-stage and how the parasite modulates the host cells in order to scavenge this essential amino acid is still unknown. The first step of methionine cycle is the synthesis of S-adenosylmethionine (SAMe) through a reaction catalyzed by the enzyme SAMe synthetase (SAMS). SAMe is a key metabolite in the methionine metabolism being the main biological donor of methyl groups for transmethylation reactions. SAMe is also a key intermediate in the transsulfuration pathway generating homocysteine (Hcy) which is metabolized into glutathione (GSH), being the last step of this pathway catalysed by glutathione synthetase (GS). GSH is a powerful antioxidant that in Plasmodium acts as one of the primary lines of the defense against the damage caused by reactive oxygen species (ROS), ensuring parasite survival. In this work we have explored the role of Plasmodium enzymes responsible for SAMe and GSH synthesis throughout its life cycle and in particular during the liver-stage of infection. The liver is a particular organ in the metabolism of methionine, namely in SAMe-dependent transmethylation reactions and in glutathione synthesis and storage. Thus, we hypothesized that while replicating inside hepatocytes, Plasmodium relies on its host to ensure a sufficient supply of these crucial metabolites. The data obtained in this study suggest that: 1) Plasmodium does not rely on its own SAMS enzyme while developing inside hepatocytes; 2) that the inhibition of SAMS activity during the blood-stage of infection leads to a low parasitemia, preventing the onset of cerebral malaria and 3) the deletion of GS-encoding gene results in the arrest at the oocyst stage, preventing transmission between the mosquito vector and the mammalian host. A detailed knowledge of Plasmodium methionine pathway provides promising tools for the design and development of novel antimalarial drugs.
A malária é uma doença causada por parasitas protozoários pertencentes ao género Plasmodium que são transmitidos aos humanos por mosquitos fêmea do género Anopheles. Apesar dos inúmeros esforços realizados na tentativa de erradicar a malária esta permanece ainda uma das doenças parasíticas mais prevalentes, constituindo um problema de saúde público. Os anti-maláricos disponíveis são insuficientes no controlo e erradicação da malária, devido sobretudo ao aparecimento de parasitas resistentes. Além disso, o escasso conhecimento acerca da biologia do parasita bem como das interações que este estabelece com o hospedeiro constituem uma barreira na luta contra a malária. Assim, o desenvolvimento de novas estratégias de intervenção torna-se crucial para conseguir a erradicação. Plasmodium é um patogénio intracelular obrigatório e, como tal, as interações que estabelece com o seu hospedeiro são essenciais para garantir o seu desenvolvimento e sobrevivência, nomeadamente as que estabelece ao nível do metabolismo da metionina. A metionina é um aminoácido essencial pelo que, tal como na maioria dos organismos, Plasmodium não tem capacidade para a sintetizar de novo. Durante a fase sanguínea Plasmodium obtém metionina maioritariamente através da degradação de hemoglobina. Contudo, os mecanismos que Plasmodium utiliza para obter metionina durante a fase hepática, bem como para modular a célula hospedeira de modo a garantir um fornecimento suficiente deste aminoácido são ainda desconhecidos. O primeiro passo do ciclo da metionina consiste na síntese de S-adenosilmetionina (SAMe) numa reação catalisada pela enzima SAMe sintetase (SAMS). A SAMe é um metabolito essencial na via metabólica da metionina sendo o maior dador biológico de grupos metilo. A SAMe é ainda um importante intermediário na via da transsulfuração sendo convertida em homocisteína e subsequentemente metabolizada em glutationo, sendo o último passo desta via catalisado pela glutationo sintetase (GS). O glutationo é um antioxidante que em Plasmodium atua como uma das primeiras linhas de defesa contra espécies oxidativas. Neste trabalho explorámos o papel das enzimas de Plasmodium responsáveis pela síntese de SAMe e glutationo ao longo do seu ciclo de vida, com particular ênfase na fase hepática da infeção. O fígado tem um papel preponderante no metabolismo da metionina, nomeadamente nas reações de transmetilação dependentes de SAMe bem como na regulação da síntese e armazenamento do glutationo. Assim, a hipótese que propusemos testar é que enquanto replica no interior do hepatócito Plasmodium depende do hospedeiro para garantir a obtenção destes metabolitos essenciais. Os resultados obtidos neste estudo demonstram que: 1) durante o seu desenvolvimento no fígado Plasmodium não depende da atividade da sua enzima SAMS; 2) a inibição da atividade da enzima SAMS durante a fase sanguínea da infeção resulta numa redução da parasitémia, prevenindo o aparecimento de malária cerebral e ainda que; 3) a deleção do gene que codifica para a enzima GS inibe o desenvolvimento dos esporozoítos, bloqueando assim a transmissão entre o vetor e o hospedeiro mamífero. Assim, um conhecimento detalhado do metabolismo da metionina em Plasmodium fornece ferramentas promissoras para o desenvolvimento de novos anti-maláricos.

博士
長庚大學
生物醫學研究所
102
As an essential amino acid, methionine plays crucial roles in multiple cellular processes. Except from diet, methionine contents are controlled by several pathways, including the folate pathway, transmethylation pathway (Methyl cycle) and methionine salvage pathway (MTA cycle). The MTA cycle is highly conserved from prokaryote to eukaryote and is distributed to regenerate methionine. However, the role of MTA cycle in physiological function is still unclear. Here, we employed MTA cycle enzyme, Drosophila aci-reductone dioxygenase 1 (DADI1), to investigate MTA cycle function in vivo. Our data demonstrated DADI1 is involved in methionine metabolism by targeted metabolites analysis. A significant decrease of methionine contents in Dadi1 mutants were found, indicating that the MTA cycle and Methyl cycle may contribute equally to regulating the level of methionine in Drosophila ovaries. Moreover, S-adenosylmethionine (SAM) and Methionine sulfoxide were also dramatic reduced, indicating that methionine related metabolites may be directly altered by MTA cycle. Furthermore, we link the MTA cycle to morphogenesis in Drosophila. The data showed that DADI1 was required for adhesion molecule Fasciclin III (FasIII) membrane expression in dorsal follicle cells and involved in dorsal appendage (DA) tubulogenesis. The defects in Dadi1 mutant cells were rescued by expressing wild-type DADI1 and human ADI1 but not ADI1 enzyme-dead mutant. Moreover, DADI1 showed genetic interaction with MTA cycle enzymes to participate in the regulation of FasIII membrane accumulation. Disrupting sam synthetase (sam-s) mimicked the effects of Dadi1 mutant in FasIII expression. Using targeted metabolites analysis, the levels of SAM were decreased substantially in sam-s mutant. In contrast, the levels of methionine increased in sam-s mutant. Supplying methionine recovered FasIII and DA defect in Dadi1 mutant but not in sam-s mutant egg chambers indicated SAM is key metabolite in regulation of FasIII expression. In addition, we revealed that SAM contents modulate the COPI mediated protein trafficking. Dadi1 and sam-s genetically interacted with COPI subunits to control FasIII expression. Knockdown COPI subunit, γCOP, by RNAi in follicle cells also caused a reduction in FasIII expression. An impairment of αCOPI signals were observed in Dadi1 mutant and sam-s mutant cells. Golgi morphology was significantly altered in Dadi1 mutant and sam-s mutant cells. Using density gradient fraction analysis, the distribution of αCOP was affected in Dadi1 mutant ovary. The protein methylation in Dadi1 mutant ovary was reduced, and a methylation inhibitor reduced FasIII expression. Together, our data demonstrate that the content of SAM may regulate membrane protein trafficking through the Golgi by controlling protein methylation, and the MTA cycle contributes to morphogenesis during development by modulating SAM contents.

Directed evolution has been explored for a long time. Various ideas, methods, have been shown to be feasible and successful in the enzyme field. We were interested in evolving enzymes for applications. Therefore, we evolved human cystathionine gamma-lyase (hCGL) and E. coli biotin ligase for therapeutic and biotechnology applications. Wild-type human cystathionine gamma-lyase does not have any methionine-degrading activity, unlike the high methionine-degrading abilities of bacterial methionine gamma-lyase (MGL) found in Pseudomonas putida. The ability to engineer hCGL to breakdown methionine can be a potential cancer treatment by targeting the methionine-dependent cancer cells. However, the methionine-degrading activity of previously engineered hCGL has only shown 1% activity compared to MGL, too low to be useful in practical cancer therapeutics. By using a combination of protein design and phylogenetic analysis, we further evolved hCGL to achieve a higher methionine-degrading activity, with one variant displaying as much as 7% activity compared to bacterial MGL, making it a more likely candidate in cancer treatment.In addition, it has been shown that new orthogonal pairs of biotin protein ligase and biotin have many biotechnology applications. Therefore, we have developed selection scheme for directing the evolution of E. coli biotin protein ligase (BPL, gene: BirA) via in vitro compartmentalization, and have altered the substrate specificity of BPL towards the utilization of the biotin analogue desthiobiotin. Following just 6 rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA₆₋₄₀ and BirA₆₋₄₇, showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. Further characterization of BirA₆₋₄₀ and the single substitution variant BirA[subscript M157T] revealed that they had 2- to 3-fold higher kcat values for desthiobiotin, and 3- to 4-fold higher K[subscript M] values. The k[subscript cat]/K[subscript M] values for these enzymes were around 0.7-fold that of BirA[subscript wt-]. It is interesting the selections did not lower the K[subscript M] for desthiobiotin and actually led to a less efficient enzyme. This is an example of how "you get what you select for". Because peptide:DNA conjugates were distributed such that there was on average one template or less per emulsion compartment there was selection only for the catalytic rate (k[subscript cat]) of desthiobiotinylation and not for turnover. Given these conditions, it might be anticipated that the peptide substrate, rather than desthiobiotin, should be bound better by the winning variants, and in fact BirA₆₋₄₀ showed a reduced K[subscript M] value for BAP.
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Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine.
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Many cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic.
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Methionine is involved in many cellular processes, several of which produce a feedback inhibitor. 5-methylthioribose (MTR) kinase, one protein involved in the removal of this inhibitor, has a protein kinase fold with conserved kinase motifs and several unique MTR binding motifs. Site-directed mutagenesis and characterization of the Bacillus subtilis enzyme was performed to probe the role of one motif. Active site D233 mutants show an activity profile similar to other protein kinase-like enzymes, suggesting a common mechanism that does not require a catalytic acid. An ordered sequential binding mechanism, with nucleotide binding first, was seen in wild type MTR kinase. Binding studies of the mutant proteins suggest that hydrogen bonding is important for MTR binding. The structures of the mutant proteins also show more differences in MTR binding than nucleotide binding. Overall, D233 is important for increasing the nucleophilicity of MTR, and ensuring its correct position in the active site.

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Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10 -3 M H 2O 2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10 -3M H 2O 2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H 2O 2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo (n = 6) and healthy controls (n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H 2O 2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed L-phenylalanine-uptake in the epidermis of acute vitiligo.