DNA adenine methyltransferase (Dam) plays an important role in different bacterial functions. It has been shown that Dam is required for regulation of bacterial replication initiation and is required for proofreading newly synthesized DNA through methylation directed mismatch repair. Dam is also involved in the regulation of different genes and is required for virulence in several different bacterial genera though its degree of importance depends on the specific bacteria being studied. During this work, a Dam-negative strain (JSM1) was constructed in Klebsiella pneumoniae strain 43816 to ascertain its importance for K. pneumoniae viability and virulence. To test JSM1 for expression of fimbrial virulence factors, agglutinations were used to detect the presence of type three and type one fimbriae, respectively. No differences between 43816 and JSM1 were discernable. Similarly, JSM1 production of capsular material appeared to be unaltered. K. pneumoniae JSM1 virulence in a murine model was examined following intranasal or intraperitoneal inoculation, and it was determined that JSM1 is partially attenuated. Quantitative analysis of 43816 and JSM1 biofilm growth revealed only slight decreases in JSM1 biofilm mass and thickness, but live/dead staining of developed biofilms showed decreased JSM1 biofilm viability over time compared to 43816 biofilms. JSM1 was also examined for alterations in the frequency of spontaneous antibiotic resistance mutations and tested for increased susceptibility to various DNA damaging agents, and statistically significant differences were found for some of the spontaneous antibiotic resistance mutation frequencies tested. Fimbriae in K. pneumoniae are important virulence factors which facilitate respiratory and urinary tract infections in vivo. They also contribute to formation of biofilms which are believed to cause chronic infections and increased antibiotic resistance. Searches for homologous regions within the Klebsiella chromosome using the chaperone and usher components of E. coli type 1 fimbriae revealed five putative fimbrial gene clusters on the Klebsiella chromosome which had not been characterized. Mutations created within select gene clusters did not yield detectable deficiencies in biofilm formation or murine respiratory virulence. However, based on the multiplicity of fimbrial expression observed in Salmonella enterica serovar Typhimurium, combinational mutations may be required prior to detection of a discernable phenotype.

Das Enzym Catechol-O- Methyltransferase (COMT) spielt eine wichtige Rolle beim Abbau der Neurotransmitter Dopamin, Noradrenalin und Adrenalin. In dessen Gen befindet sich ein Polymorphismus (SNP), der einen Aminosäureaustausch von Valin zu Methionin an Position 158 der membrangebundenden Isoform bewirkt.. In früheren Studien zeigen die verschiedenen Genotypen des Polymorphismus Unterschiede in der emotionalen Verarbeitung, bei der die COMT Einfluss auf die Verarbeitung von negativen, aber nicht von positiven Stimuli zeigt. Neben emotionalen werden durch die COMT aber auch kognitive präfrontale Prozesse beeinflusst. Eine Aufmerksamkeitslenkung auf Bilder führt im Zeitfenster der EPN und LPP zu ähnlichen Effekten wie beim Betrachten emotionaler Bilder In dieser Studie sollte daher untersucht werden, ob die COMT- Effekte auf die Emotionsverarbeitung durch Aufmerksamkeitsprozesse begründet sind und diese unabhängig vom emotionalen Inhalt durch die Aufmerksamkeitsinstruktion auslösbar sind. Dafür wurden bei 48 gesunden und entweder Val/Val oder Met/Met- Homozygoten Probanden während der Präsentation von IAPS Bildern mit positiven, negativen und neutralen Bildern ein EEG abgeleitet. Und es wurde die neuronale Aktivierung bei emotionalen Stimuli, in Interaktion mit der Instruktion, die Aufmerksamkeit auf eine bestimmte emotionale Kategorie zu richten, untersucht. Dabei zeigten sich die erwarteten Emotions- und Aufmerksamkeitseffekte auf EPN und LPP. Keinen Einfluss hatte der COMT-Genotyp. Dies könnte an der Interferenz der Emotionseffekte mit kognitiven Effekten des COMT- Polymorphismus liegen. / The catechol-O-methyltransferase (COMT) is important for the degradation of dopamine and norepinephrine in the prefrontal cortex. It exists a common single nucleotide polymorphism causing a Val to Met substitution at amino acid position 158 with the Met variant displaying lower enzymatic activity. The Val variant is associated with an impaired cognitive performance but seems to provide increased emotional resilience against negative mood states. In previous studies a positive association was found between the number of Met variants and the functional brain activity for unpleasant but not for pleasant stimuli. With regard to the tonic-phasic dopamine model it is hypothesized that this increased reactivity to negative stimuli derives from deficient disengagement from negative stimuli. Processing of emotional cues is associated with similar ERP effects as observes for explicitily directed attention. The aim of this work was therefore to investigate whether the COMT- effects on emotional processing is due to a different allocation of attentional resources. 48 homozygous participants (Met/Met or Val/Val) viewed positive, neutral or negative pictures of the IAPS while recording an EEG and where asked to either count a specific stimoulus category or to simply view the stimuli. The expected effects for emotional and attentional processing was found for the EPN and LPP. No influence was found for the COMT genotype. A possible reason is the strong interaction between emotional processing and cognitive tasks regarding the COMT polymorphism.

The antibiotic fungal metabolite sinefungin is a potent inhibitor of S-adenosylmethionine-acceptor methyltransferases. Its effect on insect metabolism and especially on corpora allata farnesoic acid methyltransferase, which catalyzes the penultimate step of juvenile hormone biosynthesis, was investigated in Locusta migratoria. Injection of sinefungin results in a delay of imaginal molt and in suppression of ovary development. Isolated corpora allata are unable to synthesize juvenile hormone III in the presence of more than 1.0 mM sinefungin. In a cell-free system containing the S-adenosylmethionine-dependent farnesoic acid methyltransferase from corpora allata sinefungin is a competitive inhibitor of the synthesis of methylfarnesoate with Ki of 1 μM.

Protein arginine methyltransferase, PRMT, is a family of epigenetic enzymes that methylate arginine residues on histone and nonhistone substrates which result in a monomethylation, symmetric dimethylation or asymmetric dimethylation via the transfer of a methyl group from S-adenosyl-L-methionine (SAM). We discovered a novel interaction between two PRMT isoforms: PRMT1 interacts and methylates PRMT6. In this study site-directed mutagenesis was performed on selected arginines identified from tandem mass spectrometric analysis to investigate major methylation sites of PRMT6 by PRMT1. In combination with radiometric methyltransferase assays, we determined two major methylation sites. Methylations at these sites have significant effects on the nascent enzymatic activity of PRMT6 in H4 methylation. PRMTs have the ability to homodimerize which have been linked to methyltransferase activity. We designed dimerization inhibitors (DMIs) to further investigate the need for dimerization for enzyme activity. Preliminary results suggest that the monomeric form of PRMT1 retains methyltransferase activity comparable to that of the uninhibited PRMT1.

No description available.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 183-205).

Abstract The enzyme phenylethanolamine N-methyltransferase (PNMT) catalyses the biosynthesis of adrenaline. Although adrenaline is a significant central nervous system (CNS) neurotransmitter, and has been associated with various physiological processes such as the control of blood pressure and the neurodegeneration observed in Alzheimer’s disease, its exact role in the CNS is unclear. As part of an international collaborative effort, this project aimed to develop PNMT inhibitors suitable for probing the role of CNS adrenaline, and to generate novel drug leads. Towards the goal of developing potent and selective PNMT inhibitors, this thesis utilised three general approaches. The first approach involved classical structure-guided drug discovery using X-ray crystallography, and is described in Chapter 2. Characterisation of the PNMT pharmacophore provided results that led to a new understanding of how PNMT recognises inhibitors. Structures described in this thesis revealed a cryptic binding pocket that is only revealed on binding of inhibitors that were predicted to be too large to interact with PNMT. The findings therefore demonstrated an extraordinary degree of flexibility inherent to the PNMT binding pocket, and emphasise the need to include greater protein flexibility in inhibitor design strategies. Secondly, this thesis investigated the catalytic mechanism of PNMT, described in Chapters 3 and 4. This research characterised the binding of substrates to wild type and variant PNMT, including the physiological substrate noradrenaline, and model substrates as well as substrate-analogue inhibitors of the enzyme. PNMT catalyses the methylation of a range of substrates. However, differential substitutions to these substrates can dictate the ligand binding position and thereby determine whether methyl transfer will occur. Additionally, the results provided new lessons for the routine use of point mutations in the study of enzymes, because changes are not always simply an indication of the difference in the residue functionality. I found, for example, that single site mutations can induce large movements in enzyme. Therefore structural characterisation of enzyme variants is an important addition to kinetic studies to enable a comprehensive examination of catalytic function. Finally, I have implemented a fragment based screening (FBS) approach to the discovery of novel lead compounds that inhibit PNMT, described in Chapters 5 and 6. The FBS approach has many advantages over existing drug discovery methods including higher hit rates, higher efficiency hits, and the ability to sample a larger range of chemical space. This thesis describes the application of FBS by X-ray crystallography to PNMT. The approach was used to screen a library of 384 compounds yielding 12 novel PNMT fragment leads. Furthermore, chemical elaboration and kinetic evaluation of these hits was performed in Chapter 7. In summary, this thesis has made a significant contribution to our understanding of the chemistry, kinetics and structure of PNMT. This understanding will be important in ongoing efforts to develop potent, selective, and CNS-active inhibitors of the enzyme.

Homocysteine, an independent risk factor for cardiovascular disease, is methylated in the liver via the zinc metalloenzyme betaine-homocysteine methyltransferase (BHMT). Established assays for BHMT include a radiochemical assay, a colorometric assay, an HPLC assay and an in vivo microbiological assay. These techniques are either unsuitable for substrate specificity studies, or are unable to give kinetic measurements. BHMT was purified from liver and measured directly and kinetically by a novel ¹H-NMR spectroscopic assay. The disappearance of substrates and the formation of products are monitored simultaneously. Using 2 mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH = 7.5) and measuring the production of N,N-dimethylglycine the CV is 6.3% (n = 6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed and had CV = 5.3%, n = 6, with a detection limit of 2 nkatal. The NMR spectroscopic assay was used to determine the substrate specificity with a library of alternative substrates. Analysis of betaine analogues with different chain length, α-substitution, substitution of the nitrogen and carboxyl moieties demonstrated that BHMT is inactive if there is any steric crowding of the nitrogen or α-carbon positions. BHMT is capable of using group VI heteroatom betaines as methyl donors, with much faster rates than glycine betaine. For glycine betaine the Km was 0.19 ± 0.03 mM with a Vmax of 17 ± 0.7 nMol min-1 mg-1. The same assay was used to detect and partially characterise a BHMT activity from hagfish liver that is similar to that of the mammalian enzyme. NMR spectroscopy was adapted for measurements of glycine betaine in urine, along with other medically significant methylamines. These were shown to be valid for clinical use and in animal studies. A novel metabolite of the sulfonium analogue of glycine betaine (methylsulfinylmethanoate) was identified in rats.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.