Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers

For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme

Methylamine as a nitrogen source for microorganisms from a coastal marine environment

Nitrogen is a key limiting resource for biomass production in the marine environment. Methylated amines, released from the degradation of osmolytes, could provide a nitrogen source for marine microbes. Thus far, studies in aquatic habitats on the utilization of methylamine, the simplest methylated amine, have mainly focussed on the fate of the carbon from this compound. Various groups of methylotrophs, microorganisms that can grow on one-carbon compounds, use methylamine as a carbon source. Non-methylotrophic microorganisms may also utilize methylamine as a nitrogen source, but little is known about their diversity, especially in the marine environment. In this proof-of-concept study, stable isotope probing (SIP) was used to identify microorganisms from a coastal environment that assimilate nitrogen from methylamine. SIP experiments using 15N methylamine combined with metagenomics and metaproteomics facilitated identification of active methylamine-utilizing Alpha- and Gammaproteobacteria. The draft genomes of two methylamine utilizers were obtained and their metabolism with respect to methylamine was examined. Both bacteria identified in these SIP experiments used the γ-glutamyl-methylamide pathway, found in both methylotrophs and non-methylotrophs, to metabolize methylamine. The utilization of 15N methylamine also led to the release of 15N ammonium that was used as nitrogen source by other microorganisms not directly using methylamine. This article is protected by copyright. All rights reserved

The Role of Protein Crystallography in Defining the Mechanisms of Biogenesis and Catalysis in Copper Amine Oxidase

Copper amine oxidases (CAOs) are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O2 to H2O2. These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer’s disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II) to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography

Využití větracích jednotek s rekuperací tepla pro nízkoenergetické domy

Import 20/04/2006Prezenční výpůjčkaVŠB - Technická univerzita Ostrava. Fakulta metalurgie a materiálového inženýrství. Katedra (635) tepelné technik

Správa majetku - údržba kolejových vozidel Dopravního podniku Ostrava

Import 19/11/2008Prezenční634 - Katedra ekonomiky a managementu v metalurgiiNeuveden

Synthesis of 1,2,4,5-tetrazine-3,6-dicarboxylic acid and its esters

Byly připraveny primární alifatické estery kyseliny dihydro-1,2,4,5-tetrazin-3,6-dikarboxylové, sloužící k přípravě esterů kyseliny 1,2,4,5-tetrazin-3,6-dikarboxylové.Katedra technologie organických látekDokončená práce s úspěšnou obhajobo

Vytápění a větrání bytových jednotek s rekuperací tepla

Import 10/10/2007Prezenční635 - Katedra tepelné technik

Maintenance System of Tram Vehicles

Import 02/11/2016Tato diplomová práce se zabývá systémem řízení údržby tramvají v Dopravním podniku Ostrava a.s., středisko vozovna Poruba. Zaměřil jsem se na průběh prováděné kontrolní prohlídky. Navrhnul jsem rekonstrukci druhé koleje na pracovišti kontrolních prohlídek, která také slouží k měření podvozků a výšky podlahy u nízkopodlažních tramvají. V oblasti provádění brzdné zkoušky jsou nově data ukládaná na záznamovou kartu tramvaje, která se vyhodnocuje pomocí počítače a záznam je uložen minimálně do další prohlídky. Vytvořená řešení zamezují zbytečnému převážení tramvaje a možnost evidence brzdné zkoušky pomocí softwarového programu.This thesis deals with the management system of maintenance of trams in Ostrava Transport Company Inc., Center depot Poruba. I focused on the progress of inspections carried out. I designed the reconstruction of the second track on the site of inspections, which also serves to measure the chassis and floor height for low-floor trams. In the implementation of the braking test the new data to be stored on the record card tram, which is evaluated using a computer and the record is saved at least until the next inspection. Create a solution avoiding unnecessary transporting trams and the possibility of evidence braking test using a software program.634 - Katedra ekonomiky a managementu v metalurgiidobř