Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Marjojen flavonoidien uuttaminen

Flavonoidit ovat kasvien polyfenolisia sekundäärimetaboliitteja, jotka koostuvat 15-hiilisestä C6-C3-C6-perusrungosta. Niillä on havaittu useita terveyden kannalta edullisia ominaisuuksia kuten antioksidatiivisia, antikarsinogeenisia, antiallergeenisia, anti-inflammatorisia, estrogeenisia sekä antimikrobiaalisia vaikutuksia. Kasvimateriaalia käyttävän elintarviketeollisuuden sivuvirrat ovat flavonoidi-pitoisten jakeiden arvokkaita raaka-aineita. Tässä työssä tarkoituksena oli tutkia marjojen puristusjäännöksen sisältämien flavonoidien uuttamista ja uuttautumiseen vaikuttavia tekijöitä kuten liuottimen koostumusta ja määrää, uuttolämpötilaa ja -aikaa sekä liuottimen lisäaineita. Uutteet analysointiin HPLC:lla käyttäen ODS-2-kolonnia ja gradienttieluointia. Komponentit detektoitiin niille tyypillisillä UV-VIS-aallonpituuksilla ja identifioitiin vertaamalla flavonoidiprofiililtaan tunnettujen marjojen kromatogrammeihin. Antosyaanien kvantitatiiviseen analysointiin käytettiin spektrofotometrista pH-eromenetelmää ja tulokset ilmoitettiin syanidiini-3-glukosidina. Uutteiden kestävyyttä tutkittiin säilyttämällä näytteitä eri olosuhteissa sekä lisäämällä niihin uuton saantoon ja komponenttien säilyvyyteen vaikuttavia aineita. Useimmat tutkituista hapoista paransivat flavonoidien saantoa ja uutteiden säilyvyyttä, mutta esim. askorbiinihapon lisääminen nopeutti tiettyjen uutteen sisältämien flavonoidien tuhoutumista.

Ellagitannins in Finnish plant species – Characterization, distribution and oxidative activity

Ellagitannins are secondary metabolites that are produced by plants. Among other features, they are assumed to function as plants’ defensive compounds against plant-eating herbivores. This thesis focuses on a theory, which suggests that the biological activity of ellagitannins is based on their tendency to oxidize at the highly alkaline gut conditions of insect herbivores (oxidative activity). To study the biological activities of ellagitannins, a wide variety of structurally different ellagitannins were purified from different plant species by using liquid chromatographic techniques. The structures were characterized with the aid of spectrometric methods. Based on the acquired data, it was also possible to create a scheme, which enables the classification and even identification of ellagitannins from plant extracts without the need to isolate each compound for individual characterization. The biological activities of ellagitannins were determined with methods that are based on the abilities of the compounds to scavenge radicals, chelate iron ions, and on their rate of oxidation at high pH. The results showed that ellagitannins possess oxidative activities both at high and neutral pH, and that their activities depend on structure. The occurrence, distribution and content of ellagitannins in Finnish plant species were also studied. The specific ellagitannin profiles of the studied plant species were found to correlate well with their taxonomic classification.

Characterisation of Cladonia rangiferina transcriptome and genome, and the effects of dehydration and rehydration on its gene expression

Lichens are symbiotic organisms, which consist of the fungal partner and the photosynthetic partner, which can be either an alga or a cyanobacterium. In some lichen species the symbiosis is tripartite, where the relationship includes both an alga and a cyanobacterium alongside the primary symbiont, fungus. The lichen symbiosis is an evolutionarily old adaptation to life on land and many extant fungal species have evolved from lichenised ancestors. Lichens inhabit a wide range of habitats and are capable of living in harsh environments and on nutrient poor substrates, such as bare rocks, often enduring frequent cycles of drying and wetting. Most lichen species are desiccation tolerant, and they can survive long periods of dehydration, but can rapidly resume photosynthesis upon rehydration. The molecular mechanisms behind lichen desiccation tolerance are still largely uncharacterised and little information is available for any lichen species at the genomic or transcriptomic level. The emergence of the high-throughput next generation sequencing (NGS) technologies and the subsequent decrease in the cost of sequencing new genomes and transcriptomes has enabled non-model organism research on the whole genome level. In this doctoral work the transcriptome and genome of the grey reindeer lichen, Cladonia rangiferina, were sequenced, de novo assembled and characterised using NGS and traditional expressed sequence tag (EST) technologies. RNA extraction methods were optimised to improve the yield and quality of RNA extracted from lichen tissue. The effects of rehydration and desiccation on C. rangiferina gene expression on whole transcriptome level were studied and the most differentially expressed genes were identified. The secondary metabolites present in C. rangiferina decreased the quality – integrity, optical characteristics and utility for sensitive molecular biological applications – of the extracted RNA requiring an optimised RNA extraction method for isolating sufficient quantities of high-quality RNA from lichen tissue in a time- and cost-efficient manner. The de novo assembly of the transcriptome of C. rangiferina was used to produce a set of contiguous unigene sequences that were used to investigate the biological functions and pathways active in a hydrated lichen thallus. The de novo assembly of the genome yielded an assembly containing mostly genes derived from the fungal partner. The assembly was of sufficient quality, in size similar to other lichen-forming fungal genomes and included most of the core eukaryotic genes. Differences in gene expression were detected in all studied stages of desiccation and rehydration, but the largest changes occurred during the early stages of rehydration. The most differentially expressed genes did not have any annotations, making them potentially lichen-specific genes, but several genes known to participate in environmental stress tolerance in other organisms were also identified as differentially expressed.

Understanding the bioactivity of plant tannins: developments in analysis methods and structure-activity studies

Tannins, typically segregated into two major groups, the hydrolyzable tannins (HTs) and the proanthocyanidins (PAs), are plant polyphenolic secondary metabolites found throughout the plant kingdom. On one hand, tannins may cause harmful nutritional effects on herbivores, for example insects, and hence they work as plants’ defense against plant-eating animals. On the other hand, they may affect positively some herbivores, such as mammals, for example by their antioxidant, antimicrobial, anti-inflammatory or anticarcinogenic activities. This thesis focuses on understanding the bioactivity of plant tannins, their anthelmintic properties and the tools used for the qualitative and quantitative analysis of this endless source of structural diversity. The first part of the experimental work focused on the development of ultra-high performance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) based methods for the rapid fingerprint analysis of bioactive polyphenols, especially tannins. In the second part of the experimental work the in vitro activity of isolated and purified HTs and their hydrolysis product, gallic acid, was tested against egg hatching and larval motility of two larval developmental stages, L1 and L2, of a common ruminant gastrointestinal parasite, Haemonchus contortus. The results indicated clear relationships between the HT structure and the anthelmintic activity. The activity of the studied compounds depended on many structural features, including size, functional groups present in the structure, and the structural rigidness. To further understand tannin bioactivity on a molecular level, the interaction between bovine serum albumin (BSA), and seven HTs and epigallocatechin gallate was examined. The objective was to define the effect of pH on the formation on tannin–protein complexes and to evaluate the stability of the formed complexes by gel electrophoresis and MALDI-TOF-MS. The results indicated that more basic pH values had a stabilizing effect on the tannin–protein complexes and that the tannin oxidative activity was directly linked with their tendency to form covalently stabilized complexes with BSA at increased pH.

Imaging Neuroinflammation in Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system CNS), where inflammation and neurodegeneration lead to irreversible neuronal damage. In MS, a dysfunctional immune system causes auto‐reactive lymphocytes to migrate into CNS where they initiate an inflammatory cascade leading to focal demyelination, axonal degeneration and neuronal loss. One of the hallmarks of neuronal injury and neuroinflammation is the activation of microglia. Activated microglia are found not only in the focal inflammatory lesions, but also diffusely in the normal‐appearing white matter (NAWM), especially in progressive MS. The purine base, adenosine is a ubiquitous neuromodulator in the CNS and also participates in the regulation of inflammation. The effect of adenosine mediated via adenosine A2A receptors has been linked to microglial activation, whereas modulating A2A receptors may exert neuroprotective effects. In the majority of patients, MS presents with a relapsing disease course, later advancing to a progressive phase characterised by a worsening, irreversible disability. Disease modifying treatments can reduce the severity and progression in relapsing MS, but no efficient treatment exists for progressive MS. The aim of this research was to investigate the prevalence of adenosine A2A receptors and activated microglia in progressive MS by using in vivo positron emission tomography (PET) imaging and [11C]TMSX and [11C](R)‐PK11195 radioligands. Magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) was performed to evaluate structural brain damage. Non‐invasive input function methods were also developed for the analyses of [11C]TMSX PET data. Finally, histopathological correlates of [11C](R)‐PK11195 radioligand binding related to chronic MS lesions were investigated in post‐mortem samples of progressive MS brain using autoradiography and immunohistochemistry. [11C]TMSX binding to A2A receptors was increased in NAWM of secondary progressive MS (SPMS) patients when compared to healthy controls, and this correlated to more severe atrophy in MRI and white matter disintegration (reduced fractional anisotropy, FA) in DTI. The non‐invasive input function methods appeared as feasible options for brain [11C]TMSX images obviating arterial blood sampling. [11C](R)‐PK11195 uptake was increased in the NAWM of SPMS patients when compared to patients with relapsing MS and healthy controls. Higher [11C](R)‐PK11195 binding in NAWM and total perilesional area of T1 hypointense lesions was associated with more severe clinical disability, increased brain atrophy, higher lesion load and reduced FA in NAWM in the MS patients. In autoradiography, increased perilesional [11C](R)‐PK11195 uptake was associated with increased microglial activation identified using immunohistochemistry. In conclusion, brain [11C]TMSX PET imaging holds promise in the evaluation of diffuse neuroinflammation in progressive MS. Being a marker of microglial activation, [11C](R)‐ PK11195 PET imaging could possibly be used as a surrogate biomarker in the evaluation of the neuroinflammatory burden and clinical disease severity in progressive MS.