Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-07T11:17:14Z (GMT). No. of bitstreams: 1 Pinheiro_Lucimar_D.pdf: 3524874 bytes, checksum: 8abbae35ad9ff5f54218f4dac80a18bf (MD5) Previous issue date: 2006 / Resumo: A utilização de enzimas para a transformação de compostos orgânicos é cada vez mais utilizada como alternativa à síntese clássica. As enzimas são utilizadas como biocatalisadores para as sínteses in vitro de compostos assimétricos uma vez que elas são intrinsicamente quirais e apresentam alta eficiência catalítica. Diante da biodiversidade de microrganismos existentes na natureza e da necessidade de descobrir novos biocatalisadores para as sínteses de blocos de construções quirais e de produtos químicos de alto valor agregado, esta tese teve como objetivos a avaliação enzimática de células microbianas íntegras, o isolamento e a identificação de compostos enantiomericamente puros obtidos através da ação enzimática de oxidorredutases. Inicialmente, a avaliação da presença de Baeyer-Villiger monooxigenases em 12 espécies de fungos através de biocatálise convencional levou a produção de (R)-(+)-5-metil-e-caprolactona (1b), produzida pelos fungos Aspergillus oryzae CCT 0975 e Geotricum candidum CCT 1205 em excelentes conversões (ambas 98%) e excessos enantioméricos (96% e 91%), respectivamente. Na etapa seguinte foi desenvolvida uma metodologia alternativa a biocatálise convencional, denominada "multibiorreações", objetivando uma triagem mais rápida e eficiente. A metodologia foi aplicada na detecção de atividade de monooxigenase permitindo um aumento no conhecimento do perfil de seletividade dos substratos analisados. A ação enzimática das células íntegras de Trichosporum cutaneum CCT 1903 resultou na redução de metilcicloexanonas orto- e para-substituídas (1 e 6) e na oxidação da cis-jasmona (8). Posteriormente realizou-se o isolamento, identificação, determinação dos excessos enantioméricos, determinação das configurações relativas e absolutas dos produtos obtidos a partir da oxidação da cis-jasmona (8): (7S,8R)-epoxijasmona, 12 (92% e.e.), 7,8-diidróxijasmona, 13 (53% e.e.) e (4S)-hidroxijasmona (86% e.e.). Nesta etapa, a determinação do perfil de seletividade de T. cutaneum CCT 1903 foi avaliado frente a 14 substratos contendo ligações duplas olefínicas (24-37). A atividade de monooxigenase foi verificada sobre os seguintes monoterpenos monocíclicos: (R)-(-)-carvona (25), a- e b- iononas (26 e 27) e (R)-(+)-limoneno (32). As biotransformações destes compostos de fragrâncias levaram às sínteses de: (1S,2R,4R)-neoisodiidrocarveol (41), (6R)-isoprenil- (3R)-metil-2-oxo-oxepanona (42), ácido-(3R)-isopropenil-6-oxo-heptanóico (43), 2,3-epóxi-(5R)-isopropenil-2-metilcicloexanol (44), 4-oxo-7,8-diidro-b-ionona (50), a-homociclogeraniol (51), limoneno-1,2-diol (54) e (+)-(4R)-p-1-menteno-8,9-diol (55), os quais foram identificados espectroscopicamente (RMN de H e C, H e H gCOSY, H e C HSQC, H e C gHMBC). Finalmente, foi realizado um estudo das atividades enzimáticas para os fungos CCT 5632, Rhyzopus oryzae CCT 1022 e a levedura AMA 7, utilizando a metodologia de multibiorreações e reações de biotransformações convencionais (substratos 8, 25-27 e 32). Uma atividade oxidorredutase foi detectada em AMA7 e R. oryzae CCT 1022. A levedura AMA7 produziu a: 7,8-epoxijasmona (12), 7,8-diidroxijasmona (13), 4- hidroxijasmona (14) e a diidrocarvona (45), enquanto que R. oryzae CCT 1022 produziu o composto 14 e o neoisodiidrocarveol (41). O fungo 5632 também apresentou atividade monooxigenase verificada a partir da formação da 4-oxo-7,8-diidro-b-ionona (50) / Abstract: The utilization of enzymes for organic compound transformations is an alternative to classical syntheses. Enzymes are used as biocatalysts for the syntheses in vitro of asymmetric compounds because they are intrinsically chiral and result in high catalytic efficiency. In front of the biodiversity of existing microorganisms in Nature and of the necessity to discover new biocatalysts for the syntheses of blocks of chiral constructions and of chemical products with high added value, this thesis aimed at enzymatic evaluation of oxidoreductase from microbial whole cells and their application of the production of enantiomerically pure compounds. First of all, Baeyer-Villiger monooxygenase (BVMO) activity was monitored using traditional biocatalytic methods. Bioprospection in 14 fungi resulted in the detection of cyclohexanone BVMO in Aspergillus oryzae CCT 0975 and Geotrichium candidum CCT 1205. The lactone (R)-(+)-1b was obtained in high enantiomeric excesses (96% and 91%, respectively) and conversion (98%). Searching for rapid screening method, multibioreaction methodology was implemented and applied to the detection of monooxigenase activity, which increased n times the amount of evaluated microorganisms per unit of time, where n is the number of added substrates. Trichosporum cutaneum CCT 1903 produced outstanding results, reducing the ortho- and para-substituted (1 and 6) methyl-cyclohexanones and oxidizing cis-jasmone (8). After isolation, identification and determination of the enantiomeric excess, the relative and absolute configuration of the cis-jasmone bioproducts were: (7S,8R)-epoxyjasmone, 12 (e.e. 92%), 7,8-dihydroxyjasmone, 13 (e.e. 53%) and (4S)-hydroxyjasmone, 14 (e.e. 86%). The enantioselectivity and substrate specificity of alkene monooxygenase in T. cutaneum CCT 1903 was further investigated using 14 substrates (24-37), applying the multibioreaction approach. Monooxygenase activity was detected in (R)-(-)-carvone, a- and b-ionones and (R)-(+)-limonene. Batch reactions of these fragrance compounds produced: (1S,2R,4R)- neoisodihydrocarveol (41), (6R)-isoprenyl-(3R)-methyl-2-oxo-oxepanone (42), (3R)- isopropenyl-6-oxoheptanoic acid (43), 2,3-epoxy-(5R)-isopropenyl-2-methylcyclohexenol (44), 4-oxo-7,8-dihydro-b-ionone (50), a-homo-cyclogeraniol (51), (R)-(+)-limonene-1,2-diol (54) and uroterpenol (55) as pure samples for spectroscopic identification (H and C NMR, H and H gCOSY, H and C HSQC, H and C gHMBC). Oxidoreductase activity was monitored using multibioreaction methodology and traditional biocatalytic methods with the fungi CCT 5632, Rhyzopus oryzae CCT 1022 and the yeast AMA 7 (substrates 8, 25-27 and 32). Thus AMA7 produced epoxyjasmone (12), 7,8- dihydroxyjasmone (13), hydroxyjasmone (14) and dihydrocarvone (45), while that R. oryzae CCT 1022 produced 14 and neoisodihydrocarveol (41). The fungus 5632 also presented monooxygenase activity confirmed through formation from 4-oxo-7,8-dihydro-b- ionone (50) / Doutorado / Quimica Organica / Doutor em Ciências

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 122-137). Also available on the World Wide Web.

In sea urchin development, cell fate specification appears by the 60-cell stage embryo when several embryonic territories are recognized: the small micromeres, the large micromeres which will generate primary mesenchyme cells, the vegetal2 layer that will give rise to pigment cells, immunocytes, and muscle cells, the vegetal1 layer, as well as the oral and aboral ectoderm. A Delta-Notch signaling event is required for the differential specification of mesodermal cells that will give rise to secondary mesenchyme cells (SMCs). SMCs produce four cell types: pigment cells, blastocoelar cells, circumesophageal muscle cells, and coelomic pouch cells. Pigment cells are the first to be specified. During primary invagination at the gastrula stage, eight pigment cell progenitors delaminate from the archenteron into the blastocoel. By the pluteus stage, approximately 30 pigment cells are embedded in the ectoderm. Pigment cells produce echinochrome, a napthoquinone pigment. Previously, several genes in the sea urchin embryo were isolated that are expressed specifically in pigment cell precursors during the blastula stage. The goal of this research was to characterize a subset of these genes, which are highly similar to: the polyketide synthase gene (Pks), a sulfotransferase gene (Sult), three different members of the flavin-containing monooxygenase gene family (Fmo), and the transcription factor glial cells missing (Gcm). Polyketide synthases (PKSs) are a large family of multifunctional proteins mainly found in bacteria, fungi, and plants. They are responsible for the biosynthesis of a variety of polyketide compounds including antibiotics and mycotoxins. In the sea urchin, SpPks is required for echinochrome biosynthesis. Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoproteins mainly found in bacteria, plants, and higher metazoan. They are responsible for catalyzing the oxidation of several compounds including the detoxification of xenobiotics and activation of numerous metabolites. It is known that SpFmo1 is required for echinochrome biosynthesis. Sulfotransferases are found from bacteria through higher eukaryotes. These enzymes catalyze the sulfate conjugation of several substrates resulting in either compound detoxification or bioactivation.

SidA from Aspergillus fumigatus is an N-hydroxylating monooxygenase that catalyzes the committed step in siderophore biosynthesis. This gene is essential for virulence making it an excellent drug target. In order to design an inhibitor against SidA a greater understanding of the mechanism and structure is needed. We have determined the crystal structure of SidA in complex with NADP+, Ornithine, and FAD at 1.9 ? resolution. The crystal structure has provided insight into substrate and coenzyme selectivity as well as residues essential for catalysis. In particular, we have chosen to study the interactions of Arg 279, shown to interact with the 2'phosphate of the adenine moiety of NADP+ as well as the adenine ring itself. The mutation of this residue to alanine makes the enzyme have little to no selectivity between coenzymes NADPH and NADH which supports the importance of the ionic interaction between Arg279 and the 2'phosphate. Additionally, the mutant enzyme is significantly more uncoupled than WT enzyme with NADPH. We see that the interactions of the guanadinyl group of Arg279 and the adenine ring are also important because KM and Kd values for the mutant enzyme are shifted well above those of wild type with coenzyme NADH. The data is further supported by studies on the reductive and oxidative half reactions. We have also explored the allosteric effect of L-arginine. We provide evidence that an enzyme/coenzyme/L-arginine complex is formed which improves coupling, oxygen reactivity, and reduction in SidA; however more work is needed to fully understand the role of L-arginine as an allosteric effector. / Master of Science in Life Sciences

Kynurenine 3-monooxygenase (K3MO) lies on the kynurenine pathway, the major pathway for the catabolism of L-tryptophan. It converts kynurenine to 3-hydroxy kynurenine. Inhibition of K3MO is important in several neurological diseases and there is evidence that inhibition of K3MO could also be targeted for the prevention of multiple organ failure, secondary to acute pancreatitis. A structure activity relationship based upon the 1,2,4-oxadiazoles motif was carried out which revealed amide 207 as an inhibitor of P. fluorescens K3MO. Further structure activity relationships were developed based upon 207. This revealed 3,4-dichloro substitution in 235 and 245 as optimum for inhibition. Co-crystalisation of these inhibitors with P. fluorescens K3MO revealed their interactions with the enzyme. It also highlighted new, potential interactions between the inhibitors and K3MO. This led to the synthesis of 271 and 272, which were also potent inhibitors of K3MO. These amides were successfully co-crystalised with P. fluorescens K3MO. Further development of the amides followed, with amide 282 providing the most potent inhibitor of P. fluorescens K3MO to date (Kᵢ = 29.1 nM).

Gilvocarcin V (GV) belongs to the angucycline class of antibiotics that possesses remarkable anticancer and antibacterial activities with low toxicity. Gilvocarcin exhibits its light induced anticancer activity by mediating crosslinking between DNA and histone H3. When photo-activated by near-UV light, the C8 vinyl group forms a [2+2] cycloadduct with thymine residues of double stranded DNA. D-fucofuranose is considered essential for histone H3 interactions. However, the poor water solubility has rendered it difficult to develop gilvocarcin as a drug. We aim to design novel gilvocarcin analogues with improved pharmaceutical properties through chemo-enzymatic synthesis and mutasynthesis. Previous studies have characterized many biosynthetic genes encoding the gilvocarcin biosynthetic skeleton. Despite these previous findings the exact functions of many other key genes are yet to be fully understood. Prior gene inactivation and cross-feeding experiments have revealed that the first isolable tetracyclic aromatic product undergoes a series of steps involving C–C bond cleavage followed by two O-methylations, a penultimate C-glycosylation and final lactone formation in order to fully develop the gilvocarcin structure. To provide a deeper understanding of these complex biochemical transformations, three specific aims were devised: 1) synthesis of the proposed intermediate and in vitro enzyme reactions revealed GilMT and GilM’s roles in gilvocaric biosynthesis; 2) utilizing in vitro studies the enzyme responsible for the C–C bond cleavage and its substrate were determined; 3) a small series of structural analogues of the intermediate from the gilvocarcin pathway was generated via chemical synthesis and fed to the mixture of the enzymes, GilMT and GilM. These reaction mixtures were then analyzed to establish the diversity of substrates tolerated by the enzymes.

Natural products are biologically active metabolites produced by fungi, bacteria, and plants that have an extended application in pharmaceutical and chemical industries. Because of their chemical versatility, flavoenzymes are commonly involved in natural product biosynthetic pathways. This has given rise to the identification of flavoenzymes that are promising candidates for biomedical and biotechnical applications. This dissertation discusses the characterization of three flavoenzymes involved in natural product biosynthesis. The class B flavin-dependent monooxygenases S-monoooxygenase from Allium sativum (AsFMO) and N-hydroxylating monooxygenase from Streptomyces sp. XY332 (FzmM) were studied. Both enzymes perform heteroatom oxidation as part of allicin or fosfazinomycin biosynthesis respectively. AsFMO was predicted to oxidize S-allyl-L-cysteine (SAC) to alliin in allicin biosynthesis. Surprisingly, AsFMO exhibited negligible activity with SAC, and instead was highly active with allyl mercaptan and NADPH. This contradicted the initial proposal and suggested that AsFMO is involved in an alternative path producing allicin directly from allyl mercaptan. FzmM was identified to perform multiple N-oxidations which lead to the formation of a nitro group. FzmM performed a highly coupled and specific reaction with L-aspartate and NADPH to produce nitrosuccinate. Both AsFMO and FzmM followed a kinetic mechanism representative of class B flavin-dependent monooxygenases with a rapid pro-R stereospecific reduction and the formation of a C(4a)-hydroperoxyflavin intermediate during oxidation. In addition, the AsFMO structure was obtained and consisted of two domains for FAD and NADPH binding signature of class B monooxygenases. The biochemical and structural study of the Acinetobacter baumannii siderophore interacting protein (BauF) was also accomplished. This enzyme is essential in acinetobactin mediated iron assimilation and is important for virulence. The characterization of the binding and reduction of acinetobactin-ferric iron complex revealed that BauF is specific for this substrate and does not utilize NAD(P)H as an electron donor. The unique activity and structure of BauF can aid future drug design. / Doctor of Philosophy / Plants, fungi, and bacteria synthesize and excrete unique chemicals called secondary metabolites or natural products. These compounds are used for many applications including dyes, flavorings, fragrances, and medicine. To make natural products, organisms use enzymes to perform complex reactions. Studying the enzymes that are involved in natural product pathways is important for understanding how secondary metabolites are made. Additionally, these enzymes can be engineered to perform reactions relevant to biotechnical applications. Our lab specializes in the study of flavoenzymes which use flavin chemistry for catalysis. Flavin is a yellow coenzyme that contributes to wide array of reactions by performing 1 or 2 electron transfers. This dissertation discussed the characterization of three flavoenzymes. The first enzyme is a S- monooxygenase from Allium sativum (garlic) called AsFMO. Reported here is the kinetic and structural characterization of AsFMO. We demonstrated that AsFMO was cabable of performing an unexpected reaction with allyl mercaptan likely converting it into allicin, the main flavor ingredient of garlic. Secondly, we reported the kinetic characterization of a nitro- forming enzyme termed FzmM. Nitro- formation is a valuable process as nitro- compounds are used in industrial organic synthesis. It was shown that FzmM performs nitro- formation with high efficiency and is specific for the substrate L-aspartate. Lastly, this work described the characterization of the the siderophore-interacting protein from Acinetobacter baumannii, BauF, which was predicted to be involved in iron acqusition. A. baumannii is a serious human pathogen with multidrug resistance, and inhibiting iron acquisition has been shown to prevent its survival. The characterization of the enzymes involved in this pathway is essential for developing new treatments for A. baumannii infection. We report the structure and function of BauF confirming its role in A. baumannii iron uptake and providing information that will aid in future drug design.

N-Hydroxylating monooxygenases (NMOs) are flavin dependent enzymes that primarily catalyze the hydroxylation of L-ornithine or L-lysine. This is the first, committed step to siderophore biosynthesis. Pathogenic microbes including Aspergillus fumigatus and Mycobacterium tuberculosis secrete these low molecular weight compounds in order to uptake FeIII from their hosts for their metabolic needs when establishing infection. Therefore, members of this family of enzymes represent novel drug targets for the development of antibiotics. Here, we present the detailed functional and structural analysis of the L-ornithine monooxygenase SidA from Aspergillus fumigatus and the L-lysine monooxygenases MbsG from Mycobacterium smegmatis and NbtG from Nocardia farcinica. The detailed chemical mechanism for flavin oxidation in SidA was elucidated for formation of the C4a-hydroperoxyflavin, deprotonation of L-ornithine, and for the chemical steps of hydrogen peroxide elimination and water elimination. This was performed through a combination of kinetic isotope effect, pH, and density functional theory studies. Also, important residues involved in substrate binding and catalysis were characterized using site-directed mutagenesis for both SidA and NbtG. These include residues involved in coenzyme selectivity, substrate binding, and residues important in C4a-hydroperoxyflavin stabilization and flavin oxidation. The kinetic mechanisms of the L-lysine monooxygenases MbsG and NbtG were characterized which show unique differences with SidA. These include differences in coenzyme selectivity, and C4a-hydroperoxyflavin stabilization. Lastly, the three-dimensional structure of NbtG was solved using X-ray crystallography which is the first structure of a lysine monooxygenase. The structure shows the NADPH-binding domain is rotated ~30° relative to the FAD-binding domain which occludes NADP+ binding in NbtG. Unlike SidA, NbtG does not stabilize a C4a-hydroperoxyflavin and this occlusion observed in the structure might explain this difference. This highlights both the structural and mechanistic diversities among NMOs and the data presented here provides valuable information for the future development of specific inhibitors of NMOs. / Ph. D.

In recent years, tyrosine kinase inhibitors have been widely used for the treatment of certain tumors as so-called targeted therapy. Many studies are concerned with their metabolism and the role of enzymes in the biotransformation process, but very little is known about the impact of tyrosine kinase inhibitors on the expression and activity of biotransformation enzymes. Nevertheless modification of the expression and activity of enzymes may cause adverse interactions of co-administered drugs and their negative impact on the human body. This diploma thesis studies the effect of tyrosine kinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes in a rat model organism in vivo. The aim was to characterize the effect of the investigated compounds on gene expression, protein expression and activity of cytochromes P450 (CYP) 1A1, 1A2 and 1B1 and flavin-containing monooxygenases FMO1 and FMO3 in renal and hepatic microsomes. Microsomes and RNA were isolated from kidneys of control rats and the pretreated rats. Western blot and immunodetection was used to compare the protein expression levels of studied enzymes in kidney and liver. By reverse transcription, cDNA was prepared from isolated RNA and used as a template for quantitative PCR to compare the...

A descoberta das monoxigenases de polissacarídeos líticas dependentes de cobre (LPMOs), que agem em sinergismo com outras enzimas na desconstrução da celulose, gerou um grande interesse da comunidade científica por seu potencial de aplicação na produção de biocombustíveis a partir de resíduos lignocelulósicos. A busca por essas proteínas auxiliares em microrganismos surgiu como uma estratégia promissora, pois há grande diversidade de isoformas e disponibilidade de sequências genômicas dessas proteínas. Diante disso, o presente trabalho teve como objetivo expressar LPMOs recombinantes de origem bacteriana em levedura Komagataella phafii. Foram obtidos clones de K. phafii transformados com 6 genes selecionados a partir de banco de dados. Análises da cinética de expressão proteica por técnica de western blot indicaram secreção apenas da LPMO de 25 kDa codificada pelo gene de Thermobifida fusca YX. Ensaios de produção da LPMO de T. fusca foram realizados em biorreator de 1L e Erlenmeyers de 250 mL e 1000 mL. Nos ensaios em Erlenmeyers houve a detecção da proteína, confirmada por SDS-PAGE e western blot, acompanhado de um alto crescimento microbiano. No ensaio em biorreator não houve detecção da proteína de interesse e o crescimento microbiano foi baixo. Com a confirmação da expressão da LPMO nos ensaios em Erlenmeyers, estes foram parcialmente purificados e avaliados por gel de proteínas e western blot. Os resultados obtidos mostram que o sistema de expressão de K. phafii foi eficiente, expressando a LPMO de T. fusca. / The discovery of copper-dependent lytic polysaccharide monooxygenase (LPMOs), auxiliary proteins which act in synergy with other enzymes on the cellulose degradation, generated a great interest from the scientific community due to their potential application in biofuels production from lignocellulosic residues. The search for these auxiliary proteins in microorganisms has emerged as a promising strategy because there is a great diversity of isoforms and availability of genomic sequences. Thus, the present work aimed to express bacterial LPMOs in Komagataella phafii. Clones of K. phafii transformed with 6 genes selected from database were obtained. Analysis of protein expression kinetics by western blot technique showed accumulation only of the LPMO of 25 kDa coded by the gene from Thermobifida fusca YX. Production of LPMO from T. fusca was tested in 1 L bioreactor and 250 mL and 1000 mL Erlenmeyer flasks. In Erlenmeyer flasks experiments, there was protein detection, confirmed by SDS-PAGE and western blot, and a high microbial growth. In the bioreactor assay, there was no target protein detection, and microbial growth was low. After confirmation of LPMO expression in Erlenmeyer flasks assays, protein were partially purified and confirmed by protein gel and western blot. Results obtained showed that the expression system of K. phafii was effective, expressing the LPMO from T. fusca.