Brodhun, Florian

[["dc.bibliographiccitation.firstpage","9052"],["dc.bibliographiccitation.issue","23"],["dc.bibliographiccitation.journal","Journal of the American Chemical Society"],["dc.bibliographiccitation.lastpage","9062"],["dc.bibliographiccitation.volume","133"],["dc.contributor.author","Fielding, Alistair J."],["dc.contributor.author","Brodhun, Florian"],["dc.contributor.author","Koch, Christian"],["dc.contributor.author","Pievo, Roberta"],["dc.contributor.author","Denysenkov, Vasyl"],["dc.contributor.author","Feussner, Ivo"],["dc.contributor.author","Bennati, Marina"],["dc.date.accessioned","2018-11-07T08:55:03Z"],["dc.date.available","2018-11-07T08:55:03Z"],["dc.date.issued","2011"],["dc.description.abstract","PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans. It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe (III) S = 5/2 high-spin (HS) and Fe(III) S = 1/2 low-spin (LS) hemes. N-14 ENDOR spectroscopy on the S = 5/2 signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = 5/2 heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T-1-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry."],["dc.description.sponsorship","DFG-IRTG [1422]; Max Planck Society"],["dc.identifier.doi","10.1021/ja202207t"],["dc.identifier.isi","000291667600049"],["dc.identifier.pmid","21548577"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/22816"],["dc.notes.status","zu prüfen"],["dc.notes.submitter","Najko"],["dc.publisher","Amer Chemical Soc"],["dc.relation.issn","0002-7863"],["dc.title","Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.peerReviewed","yes"],["dc.type.status","published"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","1449"],["dc.bibliographiccitation.issue","9"],["dc.bibliographiccitation.journal","Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids"],["dc.bibliographiccitation.lastpage","1457"],["dc.bibliographiccitation.volume","1831"],["dc.contributor.author","Koch, Christian"],["dc.contributor.author","Tria, Giancarlo"],["dc.contributor.author","Fielding, Alistair J."],["dc.contributor.author","Brodhun, Florian"],["dc.contributor.author","Valerius, Oliver"],["dc.contributor.author","Feussner, Kirstin"],["dc.contributor.author","Braus, Gerhard H."],["dc.contributor.author","Svergun, Dmitri I."],["dc.contributor.author","Bennati, Marina"],["dc.contributor.author","Feussner, Ivo"],["dc.date.accessioned","2018-11-07T09:20:44Z"],["dc.date.available","2018-11-07T09:20:44Z"],["dc.date.issued","2013"],["dc.description.abstract","In plants and mammals, oxylipins may be synthesized via multi step processes that consist of dioxygenation and isomerization of the intermediately formed hydroperoxy fatty acid. These processes are typically catalyzed by two distinct enzyme classes: dioxygenases and cytochrome P450 enzymes. In ascomycetes biosynthesis of oxylipins may proceed by a similar two-step pathway. An important difference, however, is that both enzymatic activities may be combined in a single bifunctional enzyme. These types of enzymes are named Psi-factor producing oxygenases (Ppo). Here, the spatial organization of the two domains of PpoA from Aspergillus nidulans was analyzed by small-angle X-ray scattering and the obtained data show that the enzyme exhibits a relatively flat trimeric shape. Atomic structures of the single domains were obtained by template-based structure prediction and docked into the enzyme envelope of the low resolution structure obtained by SAXS. EPR-based distance measurements between the tyrosyl radicals formed in the activated dioxygenase domain of the enzyme supported the trimeric structure obtained from SAXS and the previous assignment of Tyr374 as radical-site in PpoA. Furthermore, two phenylalanine residues in the cytochrome P450 domain were shown to modulate the specificity of hydroperoxy fatty acid rearrangement. (C) 2013 Elsevier B.V. All rights reserved."],["dc.identifier.doi","10.1016/j.bbalip.2013.06.003"],["dc.identifier.isi","000323588100003"],["dc.identifier.pmid","23797010"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/28946"],["dc.notes.status","zu prüfen"],["dc.notes.submitter","Najko"],["dc.publisher","Elsevier Science Bv"],["dc.relation.issn","1388-1981"],["dc.title","A structural model of PpoA derived from SAXS-analysis-Implications for substrate conversion"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.peerReviewed","yes"],["dc.type.status","published"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","1594"],["dc.bibliographiccitation.issue","9"],["dc.bibliographiccitation.journal","FEBS Journal"],["dc.bibliographiccitation.lastpage","1606"],["dc.bibliographiccitation.volume","279"],["dc.contributor.author","Koch, Christian"],["dc.contributor.author","Fielding, Alistair J."],["dc.contributor.author","Brodhun, Florian"],["dc.contributor.author","Bennati, Marina"],["dc.contributor.author","Feussner, Ivo"],["dc.date.accessioned","2018-11-07T09:10:52Z"],["dc.date.available","2018-11-07T09:10:52Z"],["dc.date.issued","2012"],["dc.description.abstract","Psi factor producing oxygenases (Ppos) are fusion proteins consisting of a peroxidase-like functionality in the N-terminus and a P450-fold in the C-terminal part of the polypeptide chain. It was shown that they are responsible for the production of oxidized fatty acids that play a pivotal role in the control of fungal colonization of plant and mammalian hosts. The similarity of the primary structure of the single domains to various host-derived oxylipin-forming enzymes and functional conservation of these enzymatic activities was the basis for prediction of the 3D conformations of the single domains of a prototype Ppo enzyme. We were able to predict a putative substrate binding pocket in the N-terminal domain of the enzyme and support this finding by site-directed mutagenesis. With the proposed substrate binding mode all known determinants of oxygen insertion are in a reasonable spatial arrangement for catalysis. Additionally, we could identify an arginine and show its involvement in substrate binding by kinetic analysis of the respective variant. While substrate position in the dioxygenase domain is well defined, our results indicate that the substrate binding to the P450 domain is rather unconstrained. Nevertheless an asparagine residue within the I-helix is shown to be involved in catalysis and promotes a shortcut of the typical P450 reaction cycle. Taken together, the results presented here exemplify that fatty acids are oxidized in all kingdoms of life by structural and functional highly conserved enzymes."],["dc.identifier.doi","10.1111/j.1742-4658.2011.08352.x"],["dc.identifier.isi","000302995500008"],["dc.identifier.pmid","21920024"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/26594"],["dc.notes.status","zu prüfen"],["dc.notes.submitter","Najko"],["dc.publisher","Wiley-blackwell"],["dc.relation.issn","1742-464X"],["dc.title","Linoleic acid positioning in psi factor producing oxygenase A, a fusion protein with an atypical cytochrome P450 activity"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.peerReviewed","yes"],["dc.type.status","published"],["dspace.entity.type","Publication"]]

[["dc.bibliographiccitation.firstpage","728"],["dc.bibliographiccitation.issue","5"],["dc.bibliographiccitation.journal","ChemBioChem"],["dc.bibliographiccitation.lastpage","737"],["dc.bibliographiccitation.volume","12"],["dc.contributor.author","Nadler, Andre"],["dc.contributor.author","Koch, Christian"],["dc.contributor.author","Brodhun, Florian"],["dc.contributor.author","Wehland, Jan-Dirk"],["dc.contributor.author","Tittmann, Kai"],["dc.contributor.author","Feussner, Ivo"],["dc.contributor.author","Diederichsen, Ulf"],["dc.date.accessioned","2018-11-07T08:58:03Z"],["dc.date.available","2018-11-07T08:58:03Z"],["dc.date.issued","2011"],["dc.description.abstract","PpoA is a bifunctional enzyme that catalyzes the dioxygenation of unsaturated C18 fatty acids. The products of this reaction are termed psi factors and have been shown to play a crucial role in conferring a balance between sexual and asexual spore development as well as production of secondary metabolites in the fungus Aspergillus nidulans. Studies on the reaction mechanism revealed that PpoA uses two different heme domains to catalyze two subsequent reactions. Initially, the fatty acid substrate is dioxygenated at C8, yielding an 8-hydroperoxy fatty acid at the N-terminal domain. This reaction is catalyzed by a peroxidase/dioxygenase-type domain that exhibits many similarities to prostaglandin H2 synthases and involves a stereospecific homolytic hydrogen abstraction from C8 of the substrate. The C terminus harbors a heme thiolate P450 domain in which rearrangement of the 8-hydroperoxide to the final product, a 5,8-dihydroxy fatty acid, takes place. To obtain further information about the intrinsic kinetics and reaction mechanism of PpoA, we synthesized C5-dideutero- and C8-dideutero- oleic acid by a novel protocol that offers a straightforward synthesis without employing the toxic additive hexamethylphosphoramide (HMPA) during C-C coupling reactions or mercury salts upon thioketal deprotection. These deuterated fatty acids were then employed for kinetic analysis under multiple-turnover conditions. The results indicate that the hydrogen abstraction at C8 is the rate-determining step of the overall reaction because we observed a KIE (V(H)/V(D)) of similar to 33 at substrate saturation that suggests extensive nuclear tunneling contributions for hydrogen transfer. Deuteration of the substrate at C5, however, had little effect on V(H)/V(D) but resulted in a different product pattern presumably due to an altered lifetime and partitioning of a reaction intermediate."],["dc.description.sponsorship","DFG [IRTG 1422]"],["dc.identifier.doi","10.1002/cbic.201000669"],["dc.identifier.isi","000288563000013"],["dc.identifier.pmid","21365732"],["dc.identifier.uri","https://resolver.sub.uni-goettingen.de/purl?gro-2/23552"],["dc.notes.status","zu prüfen"],["dc.notes.submitter","Najko"],["dc.publisher","Wiley-blackwell"],["dc.relation.issn","1439-4227"],["dc.title","Influence of Substrate Dideuteration on the Reaction of the Bifunctional Heme Enzyme Psi Factor Producing Oxygenase A (PpoA)"],["dc.type","journal_article"],["dc.type.internalPublication","yes"],["dc.type.peerReviewed","yes"],["dc.type.status","published"],["dspace.entity.type","Publication"]]