32 research outputs found

    Structure & Function of Enzymes in Two Uncharacterized Gene Clusters from Pseudomonas Brassicacearum & Streptomyces Griseofuscus

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    Pyridoxal 5’-phosphate (PLP)-dependent enzymes harness this versatile cofactor to catalyze a variety of reactions including transamination, decarboxylation, racemization and various elemination/subsitution reactions. Several years ago, a new class of PLP-dependent enzymes was discovered that uses PLP and molecular oxygen to catalyze the 4-electron oxidation of L-arginine to 4-hydroxy-2-ketoarginine. Work with the prototypical enzyme of this class, MppP from Streptomyces wadayamensis (SwMppP), showed that the dioxygen consumed during the reaction is reduced to hydrogen peroxide, and that the hydroxyl group installed in the product derives from water. Thus, SwMppP is an L-arginine oxidase, and not an oxygenase. This was surprising given that the hydroxylation step occurs at an un-activated methylene group of the substrate, L-arginine. SwMppP is part of the mannopeptimycin biosynthetic cluster and works together with MppR and MppQ to synthesize the non-proteinogenic amino acid L-enduracididine (L-End). A BLASTp search of the NR database using the sequence of S. wadayamensis MppP revealed close homologs in a number of species of pseudomonads, including Pseudomonas brassicacearum, P. syringae, P. amygdali, and P. aeruginosa. The first section of this thesis is focused on the genomic context of the pseudomonad MppP homologs. None of the pseudomonad genomes examined contained other key mannopeptimycin biosynthetic genes like the nonribosomal peptide synthetases mppA and mppB, or the other two L-End biosynthetic genes mppQ and mppR. The lack of MppQ and MppR homologs in these organisms suggests that the arginine oxidase activity of MppP homologs is used in a distinct biochemical context. We cloned, expressed, and purified the MppP homolog from Pseudomonas brassicacearum, a soil-dwelling bacterium associated with the roots of plants in the mustard/cabbage family that protects the plants from several bacterial and fungal pathogens. The genomic context of the gene encoding this MppP homolog (PbrMppP) consists of a regulatory gene, the MppP homolog, and four additional open reading frames annotated as hypothetical protein (PbrHYP), dihydrodipicolinate synthase family protein (PbrDHPS), Mononuclear Fe-dependent oxygenase; (PbrOX), and 2Fe2S-binding protein ferredoxin (PbrFD). All these genes were annotated from sequence analysis. The observations that (1) the genes are overlapping, (2) that the open reading frame includes a luxR-type regulatory gene, and (3) 5’-untranslated region contains a putative promoter sequence suggests that these genes may constitute an operon, which we will refer to as the P. brassicacaerum MppP containing operon (PbrMPCO). Understanding the biochemical context of PbrMppP requires assigning functions for the other 4 non-regulatory gene products. To begin, we confirmed that the structure and activity of PbrMppP are both identical to the prototypical SwMppP. Next, we analyzed the X-ray crystal structures of PbrHYP, PbrDHPS and PbrOx. We also identified condition that activates the promoter of this gene cluster. Since the structures were not sufficient to deduce the functions of these enzymes, we turned to a metabolomic strategy to determine the final product of theiv operon. Knowing the final product would limit the possibilities for substrate of these putative enzymes, we show here a comparative metabolomic analysis by knocking out the entire operon and searching for “missing” metabolite(s) by multiple MS. The second section focuses on a second MppP homolog from Streptomyces griseofuscus. The gene encoding this MppP homolog, SgrMppP, is flanked by 5 other genes whose products are annotated as a putative biotin carboxylase (SgrLIG), flavin-dependent monooxygenase (SgrOX), S-adenosyl methionine (SAM)-dependent methyltransferase (SgrMT), amidinohydrolase (SgrAH), and GNAT-family acetyltransferase (SgrNAT). The goal of these studies is to determine the activities of all the enzymes in this gene cluster and to identify the final product. So far I have shown, in the presence of Mg(II) and SAM, SgrMT catalyzes the transfer of a methyl group from SAM to carbon 4 of the 4-hydroxy-2-ketoarginine produced by SgrMppP. We also determined that SgrMT products are used by SgrAH as substrate and the turned over products by SgrAH are ornithine derivatives and urea. Succeeding research will involve structural and functional determination of rest of the gene products, which will eventually lead to the identification of final product of these gene-contexts

    Bioactive actinobacteria associated with two South African medicinal plants, Aloe ferox and Sutherlandia frutescens

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    Philosophiae Doctor - PhDActinobacteria, a Gram-positive phylum of bacteria found in both terrestrial and aquatic environments, are well-known producers of antibiotics and other bioactive compounds. The isolation of actinobacteria from unique environments has resulted in the discovery of new antibiotic compounds that can be used by the pharmaceutical industry. In this study, the fynbos biome was identified as one of these unique habitats due to its rich plant diversity that hosts over 8500 different plant species, including many medicinal plants. In this study two medicinal plants from the fynbos biome were identified as unique environments for the discovery of bioactive actinobacteria, Aloe ferox (Cape aloe) and Sutherlandia frutescens (cancer bush)

    HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE: WHEN STRUCTURE AND MOBILITY DRIVE EFFICIENT CATALYSIS. IMPLICATIONS FOR AADC DEFICIENCY

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    L’enzima Decarbossilasi degli L-amino acidi aromatici (AADC) è responsabile della sintesi di due neurotrasmettitori essenziali: la dopamina e la serotonina. AADC deve la sua attività catalitica alla chimica del suo cofattore, il piridossale 5’-fosfato (PLP). La struttura cristallografica dell’enzima da mammifero (precisamente da maiale che ha il 90% di identità con l’enzima umano) nella sua forma olo venne risolta venti anni fa e tale risoluzione aprì la strada ad importanti studi strutturali. Dieci anni dopo venne pubblicata la struttura umana di AADC nella sua forma apo evidenziando quali cambiamenti conformazionali avvengono quando il PLP viene legato dall’enzima. Le strutture apo e olo AADC hanno avuto notevole importanza per la comprensione della patogenicità di varianti enzimatiche associate alla malattia chiamata ‘Deficit da AADC’ (AADCd, OMIM#608643). Questa malattia autosomica recessiva molto rara è dovuta prevalentemente a mutazioni missenso sul gene AADC. I pazienti affetti da AADCd mostrano un’amAromatic L-Amino Acid Decarboxylase (AADC) is the enzyme responsible for the synthesis of two essential neurotransmitter dopamine and serotonin from L-Dopa and L-hydroxytryptophan. AADC owes its specific catalytic activity to the chemistry of its cofactor, pyrydoxal-5’-phosphate (PLP). Almost 20 years ago, the crystal structure of a mammalian holoAADC (porcine, sharing 90% of sequence identity) was solved and the availability of its 3D structure paved the way to structural studies. Moreover, 10 years later, human apoAADC structure was published, shedding light on the conformational rearrangement occurring on the apo enzyme upon addition of PLP. Importantly, apo and holoAADC structures provided crucial insights for the comprehension of the pathogenicity of a number of AADC deficiency associated variants. AADC deficiency (OMIM#608643) is a rare autosomal recessive inborn disease due to missense mutations in the AADC gene. Patients bearing these mutations show mild to severe phenotypes, whose destiny is often fatal. Due to the rarity of the disease and to the heterogeneous response to the treatments, medications are not often satisfactory. In the past years, some efforts on human recombinant AADC pathogenic variants have tried to provide support to the research on AADC deficiency by means of biochemical and biophysical approaches determining the impact of the amino acid substitutions on the enzyme features. Here, a further contribution to the comprehension of the AADC deficiency is provided. The crystal structure of human holoAADC has been solved under different conditions, both in its native and ligand bound form. The combination of crystallographic studies, molecular dynamics simulations (MD) and site directed mutagenesis uncovered novel aspects of the AADC structure-function relationship. Moreover, the characterization of 21 novel identified pathogenic variants (spread on each AADC domain, N-terminal, Large and C-terminal Domains) led to the widening of the range of enzymatic phenotypes associated to AADC deficiency. The proposed combination of biochemical and kinetic studies permitted to determine correlations between structural and functional signals. Enzymatic phenotypes span from variants characterized by a mild phenotypes to variants (mainly located at the NTD-CTD interface) whose dramatic structural defects lead to a catalytic incompetence. In addition, MD simulations and in solutions data point out a critical role for the loop3 element that contains the essential catalytic residue Tyr332. A group of variants affecting loop3 has been identified as catalytically incompetent and their structural features have been dissected thanks also to the solving of the crystal structure of pathogenic variant L353P, which constitutes the first solved structure of an AADC variant. Altogether, this study on human AADC provides new elements for the comprehension of the structure-function relationship of AADC with a particular focus on protein dynamics and mobility. Lastly, structural details might represent the basis for both the designing of novel specific inhibitors and for a better comprehension of the molecular aspects of the variants associated with the AADC deficiency

    INVESTIGATION OF THE BIOSYNTHESIS OF THE NUCLEOSIDE ANTIBIOTIC SPHAERIMICIN

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    Antibiotic-resistance has become a widespread problem in the United States and across the globe. Meanwhile, new antibiotics are entering the clinic at an alarmingly low rate. Highly-modified nucleosides, a class of natural products often produced by actinobacteria, target MraY bacterial translocase I. MraY is a clinically unexploited enzyme target that is ubiquitous and essential to peptidoglycan cell wall biosynthesis. The nucleoside antibiotics known vary in efficacy and the functionalities contributing to improved activity is poorly understood. Sphaerimicin, a newly discovered modified nucleoside, has potent inhibitory activity with an IC50 of 13.65 nM against MraY. In general, sphaerimicin is primarily effective against gram-positive bacteria (MIC ranges from 2-16 μg/mL against Enterococcus faecium and Staphylococcus aureus), but little is known about the biosynthesis and mechanism of action. Sphaerimicin has highly unusual structural features, including a heavily modified ribosylated glycl-uridine disaccharide core that is appended to a dihydroxylated piperidine ring. The novel biosynthesis of these features was investigated, leading to the functional characterization of six enzymes critical for the disaccharide core formation from uridine monophosphate. Recently, a unique S-adenosylmethionine- and PLP-dependent alpha-aminobutyric acid transferase and a nonheme Fe(II)- and alpha-ketogluterate-dependent hydroxylase from the sphaerimicin biosynthetic pathway were characterized. This part of the pathway extends the carbon scaffold, which will be crucial for formation of the piperidine ring. Not only does this study provide new chemical entities to help better understand MraY as a target, it could potentially reveal new enzymatic chemistries that will power innovative chemoenzymatic synthesis and genome mining to uncover new natural products

    Metabolic screening for PKS and NRPS in endophytic actinobacteria from citrus reticulata

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    Polyketides and non-ribosomal peptides are natural products widely found in bacteria, fungi and plants. The biological activities associated with these metabolites have attracted special attention in biopharmaceutical studies. Polyketide synthases act similarly to fatty acids synthetases and the whole multi-enzymatic set coordinating precursor and extending unit selection and reduction levels during chain growth. Acting in a similarly orchestrated model, non-ribosomal peptide synthetases biosynthesize NRPs. PKSs-I and NRPSs enzymatic modules and domains are collinearly organized with the parent gene sequence. This arrangement allows the use of degenerated PCR primers to amplify targeted regions in the genes corresponding to specific enzymatic domains such as ketosynthases and acyltransferases in PKSs and adenilation domains in NRPSs. Careful analysis of these short regions allows the classifying of a set of organisms according to their potential to biosynthesize PKs and NRPs. In this work, the biosynthetic potential of a set of 13 endophytic actinobacteria from Citrus reticulata for producing PKs and NRP metabolites was evaluated. The biosynthetic profile was compared to antimicrobial activity. Based on the inhibition promoted, 4 strains were considered for cluster analysis. A PKS/NRPS phylogeny was generated in order to classify some of the representative sequences throughout comparison with homologous genes. Using this approach, a molecular fingerprint was generated to help guide future studies on the most promising strains383333341CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPPQ 306932/2009-12008/00605-1; 2008/06097-8; 2009/03793- 6; 2010/16798-

    Технологія виробництва стимулятора росту рослин. Ділянка біосинтезу продукту

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    Дипломний проект містить 112 с., 14 рис., 8 табл., 73 посилання. Робота присвячена розробці технології виробництва препарату стимулятора росту рослин. Ділянка біосинтезу продукту. Запропоновано в якості продуцента комплексу гідролітичних ферментів та фітогормонів для стимуляції росту рослин використовувати штам Streptomyces albus UN44, що володіє здатністю надпродукувати цільовий продукт. В роботі обґрунтовані та подані технологічна та апаратурна схема виробництва стимулятора росту рослин. Розраховано та вибрано найбільш ефективне обладнання для біосинтезу продукту. Наведено технологічний та конструктивний розрахунки біореактора. Розроблена технологія повністю відповідає санітарно-гігієнічним та екологічним нормам, наведено техніку безпеки та охорону праці.The graduation project contains 112 pages, 14 figures, 8 tables, 73 references. The work is devoted to the development of the technology for the production of a plant growth promoter. The site of product biosynthesis. It has been proposed to use a strain of Streptomyces albus UN44, which has the ability to overproduce the target product, as a producer of a complex of hydrolytic enzymes and phytohormones to stimulate plant growth. The technological and instrumental scheme of production of plant growth stimulator production is justified and presented in the work. The most efficient equipment for product biosynthesis has been calculated and selected. Technological and constructive calculations of the bioreactor are given. The developed technology fully complies with the sanitary-hygienic and environmental standards, safety and labor protection are given

    Structure-function relationships in the MppP family

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    The rare nonproteinogenic amino acid L-enduracididine (L-End) is a building block of some naturally occurring antibiotics like mannopeptimycin. Antibiotic-resistant pathogens are an imminent threat to public health. Studying the biosynthetic pathways by which natural antibiotics are formed by bacteria and fungi can provide valuable information for drug design. L-End biosynthesis involves one particularly interesting enzyme, a pyridoxal-5’phosphate (PLP) dependent L-Arg oxidase, called MppP. MppP is unique in that it is the first PLP-dependent enzyme to perform a 4-electron oxidation with just molecular oxygen and PLP. This is highly unusual in that this sort of chemistry typically uses metals or more exotic organic cofactors. A number of MppP homologs have been identified and this small family actually contains two different groups. One catalyzes the reaction that results in the hydroxylated arginine product, while the other has a fundamentally different reaction mechanism that results in the product 4,5-dehydro-2-ketoarginine. Our studies aim to identify the structural differences between these two groups of enzymes that result in their having different catalytic mechanisms. So far, the active sites of these homologs seem to have completely conserved amino acid sequences and thus likely very similar structures. Here we describe the first complete structure of a dehydrating MppP homolog from Pseudoalteromonas luteoviolacea (PlMppP). This structure is compared to the prototypical hydroxylating MppP from Streptomyces wadayamensis (SwMppP). We also report on the expression and purification of several other dehydrating MppP homologs, the structures of which will eventually allow us to refine and extend our analysis. The structural data we are generating will have implications for adapting MppP-like enzymes to be used as biocatalysts for the synthesis of potentially novel life-saving antibiotics

    Genome Mining Revealed a High Biosynthetic Potential for Antifungal Streptomyces sp. S-2 Isolated from Black Soot

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    The increasing resistance of fungal pathogens has heightened the necessity of searching for new organisms and compounds to combat their spread. Streptomyces are bacteria that are well-known for the production of many antibiotics. To find novel antibiotic agents, researchers have turned to previously neglected and extreme environments. Here, we isolated a new strain, Streptomyces sp. S-2, for the first time, from black soot after hard coal combustion (collected from an in-use household chimney). We examined its antifungal properties against plant pathogens and against fungi that potentially pose threat to human health (Fusarium avenaceum, Aspergillus niger and the environmental isolates Trichoderma citrinoviridae Cin-9, Nigrospora oryzae sp. roseF7, and Curvularia coatesieae sp. junF9). Furthermore, we obtained the genome sequence of S-2 and examined its potential for secondary metabolites production using anti-SMASH software. The S-2 strain shows activity against all of the tested fungi. Genome mining elucidated a vast number of biosynthetic gene clusters (55), which distinguish this strain from closely related strains. The majority of the predicted clusters were assigned to non-ribosomal peptide synthetases or type 1 polyketide synthetases, groups known to produce compounds with antimicrobial activity

    A Study in Molecular Recognition: Synthesis of a Β-sheet Mimic & Quantitation of Metal Ions in Aqueous Solutions Through Solid Supported Semi-selective Chemosensors

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    From the hydrophobic effect, which is responsible for the organization of amphipathic molecules into cellular membranes, to the highly specific hydrogen binding patterns found in DNA base pairs that keeps our genetic material “zipped up”, non-covalent and reversible interactions are critical to properly functioning biological processes. Molecular recognition is an area of study that seeks to better understand these observed phenomena. In a general sense, association of “Host” and “Guest” molecules are based on ionic forces, hydrophobic interactions, cation-π effects, π-π stacking, conformational restriction, and many others. This dissertation will primarily focus on two projects that have an emphasis on studying molecular recognition. The first major project details the synthesis of a molecule that mimics the hydrogen bonding array of a β-sheet. β-sheets, secondary protein structures found ubiquitously in nature, are composed of peptide strands that associate through hydrogen bonds between an amide carbonyl on one strand to an amide -NH on an adjacent strand. As peptide strands begin to fold into a β-strand it pre-organizes the hydrogen bond donors and acceptors on the other edge allowing for the β-strand to propagate into a β-sheet. While this propagation is beneficial in the efficient folding of proteins, it makes it difficult for scientists to study this phenomenon in solution apart from the other complexities that exist in protein structures. Chemists have addressed this issue by creating synthetic mimics that simulate the hydrogen bonding array found in β-sheets along only one edge, greatly simplifying the observable phenomena and allowing them to study these effects in greater detail in solution. Based on the work of previous chemists I have developed a synthetic β-sheet mimic that can replace 3 amino acids in a peptide, has fluorescent properties, and can be incorporated by solid phase synthetic methods into peptides. Using a quinolone as a fluorescent core, I have synthesized a 3,6-diaminoquinol-4-one that has the same hydrogen bonding array. Preliminary studies appear to show association with itself in organic solvents. Additionally, I have developed synthetic schemes towards a pyrido[2,g]quinolone that would retain the same hydrogen binding array with a higher degree of conformational restriction and presumed fluorescent properties. This synthetic work will allow for future graduate students to study these hydrogen bonding interactions. The second major project in this dissertation details the work I have done on a hydrogel solid support. This work was done to enable the development of a real-time continuously monitoring sensor for the detection and quantitation of metal ions in aqueous solution. Specific azo dyes have long been known to show a shift in their absorbance spectrum with the addition of metal ions. When used as soluble molecules they are difficult to reuse due to their strong association to the metal ions. I have developed various hydrogel polymers with covalently attached azo dyes capable of metal ion diffusion in aqueous solutions. Optimization of these hydrogels has been achieved by variation of composition, crosslink-density, co-solvent selection and glass derivatization allowing for a robust attachment to a rigid backing. These hydrogels are optically transparent, allow for removal of the metals with acidic media, and demonstrate sufficient mechanical strength to allow them to be easily moved between analyte solutions. Two separate type of polymers have been developed to allow for either alkylation or acylation reactions to produce the covalent linkage of dye to hydrogel, each with its own advantages. With others in my research group and in collaboration with a local Milwaukee company, we have shown the azo-dyes covalently tethered to these hydrogels retain their optical properties and can be used for the identification and quantitation of aqueous metal species when incorporated into a flow cell. They are stable to hundreds of binding and release cycles and months of use, at least

    11th Annual Undergraduate Research Symposium

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