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Lot6p – a redox regulated switch of the proteasome During our previous studies of bacterial quinone reductases, we have also investigated the biochemical properties of the yeast homolog Lot6p. Despite the availability of a three-dimensional structure for Lot6p (1T0I), the physiological role of the enzyme was unclear. Our recent studies have now demonstrated that the enzyme rapidly reduces quinones at the expense of a reduced nicotinamide cofactor, either NADH or NADPH. In order to further characterize the cellular role of Lot6p, we have carried out pull-down assays and identified the 20S core particle of the yeast proteasome as interaction partner. Further studies revealed that this complex recruits Yap4p, a member of the b-Zip transcription factor family, but only when the flavin-cofactor of Lot6p is in its reduced state. Oxidation of the flavin leads to dissociation of the transcription factor and relocalization to the nucleus (see scheme below). reduced Lot6p α β β α A similar system is known from mammalian cells, where a homologous quinone reductase (NQO1) binds to the 20S proteasome and recruits important tumor suppressor proteins such as p53 and p73α. Hence, the discovery of a homologous protein interaction in yeast provides an interesting model system to investigate the molecular basis for protein complex formation and regulation of proteasomal degradation of transcription factors (postdoctoral project of Ines Waldner-Scott; thesis project of Sonja Sollner and Venugopal Gudipati). Zn-dependent Alkylsulfatases Yap4p Oxidation by e.g. quinones Hydrolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize sulfur. Three classes of sulfatases have been discovered, and the recently elucidated structure of SdsA1 from Pseudomonas aeruginosa revealed that the enzyme belongs to the widely occurring family of Zn-dependent ß-lactamases. A detailed characterization of the enzyme’s stereochemistry by the group of Prof. Faber at the University of Graz demonstrated that this enzyme cleaves primary alklysulfates (preferred substrate is dodecylsulfate) by retention. In other words, the hydroxyl group attacks the sulfur atom in the course of the reaction (see reaction scheme below). Only recently a novel enzyme activity could be identified in another Pseudomonas strain, which leads to hydrolytic cleavage of alkylsulfates via stereoinversion, i. e. the hydroxyl group attacks the carbon rather than the sulfur atom of the substrate (see scheme below). 10 oxidized Lot6p Very slow with oxygen O2 nucleus oxygen β α β α Yap4p Yap4p
HO R 1 H R 2 HSO 4 - Inversion H R 1 OH - O 11 R 2 SO 3 - OH - HSO 4 - Retention Despite the differences in substrate preference and stereochemistry, the overall structure of this novel enzyme (termed Pisa1 = Pseudomonas inverting sulfatase) is virtually identical to the previously determined structure of SdsA1. However, the active site of Pisa1 features several conspicuous amino acid exchanges (see figure below: in blue active side residues in SdsA1 and green those in Pisa1). Phe/Gly Tyr/His Ala/Ile These amino acids are now subject of an extensive mutagenesis program to define their role in governing substrate preference and the stereochemical course of the reaction. This project is a close collaboration with Profs. Faber (biocatalysis) and Wagner (structure determination) from the University of Graz (thesis project of Tanja Knaus in our laboratory and Markus Schober in Prof. Faber’s laboratory). Doctoral theses completed Tyr/Ser Met/Ser Met/Ser Tyr/Ser Leu/Pro Sonja Sollner: Function and mechanism of Lot6p from Saccharomyces cerevisiae, a flavindependent quinone reductase Flavin-dependent NAD(P)H:quinone oxidoreductases have been reported from several mammalian sources. These enzymes have been thought to have a protective effect against quinone-related oxidative cell damage through their unique ability to transfer two electrons to a H R 1 Ala/Ile OH R2
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Page 9: Nikkomycin biosynthesis Nikkomycins
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