Structure and function of A.nidulans PSI factor producing oxygenase A
von Christian Koch
Datum der mündl. Prüfung:2012-10-01
Erschienen:2013-04-25
Betreuer:Prof. Dr. Ivo Feußner
Gutachter:Prof. Dr. Ivo Feußner
Gutachter:Prof. Dr. Marina Bennati
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http://dx.doi.org/10.53846/goediss-3813
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Zusammenfassung
Englisch
Phytopathogenic fungi are not only of economic importance for the yield depression they cause, but can also be considered as severe threat for the health of the consumer ingesting molded crops and fruits. This health threat is caused by several fungal secondary metabolites referred to as mycotoxins (Section 1.2.1). About eight years ago, the group of Nancy Keller proposed that these molding processes, as well as several types of mycoses in mammals, are mediated by fungal hormone-like acting compounds known as Psi-factors (Section 1.2.2). Considering the economic importance of the provoked fungal infections and their impact on public health, a detailed investigation of the underlying mechanisms of Psi-factor perception, their physiological influence and biosynthesis is of scientific significance. Focusing on the biochemical and mechanistic characterization of Psi-factor producing oxygenase A and thus highlighting the molecular basis of Psi-factor biosynthesis, this study could substantiate the mechanism presented by Brodhun et al. (Brodhun et al., 2009) and establish a hypothetical structural model. Although being merely a low resolution model with atomic details based solely on structure prediction and the overall-shape based on small-angle X-ray scattering (Section 4.6.1), several aspects of this structure could be validated by the deployment of adequate biochemical and biophysical approaches. Exemplary the crucial involvement of five newly identified amino acids in determination of the reaction specificity and enzyme activity could be proven (Section 4.4). So it was shown that the fatty acid substrate may bind to the DOX-domain with the ω-end first and is stabilized by ionic interactions of its carboxylate with Arg336, while binding of the intermediately formed hydroperoxy fatty acid to the cytochrome P450-domain may occur in a reversed orientation, i.e. with the carboxyl-end first, and thus requires an uncharged, protonated substrate. Instead two phenylalanines (Phe795 and Phe799) seem to play a role in substrate-binding and determination of reaction specificity. While the rearrangement reaction catalyzed by the P450-domain involves heterolytic cleavage of a peroxide, which is facilitated by the presence of Asn887, the dioxygenation in the N-terminal domain of PpoA is mediated by a tyrosyl radical. Based on the active site structure, two tyrosines were postulated to be involved in this process. While EPR-spectroscopy of the respective variants revealed that Tyr374 ((Fielding et al., 2011) and section 4.6.2) is most likely the catalytic active residue, Tyr327 seems to be involved in orienting this residue in a catalytic competent position. Besides these active site structures guiding the enzymatic turn-over, a distinct aspect of the enzyme mechanism was evaluated by the use of specifically dideuterated substrates (Section 4.7). Thus it was shown that hydrogen abstraction preceding the insertion of molecular oxygen is the rate limiting step of the overall reaction and both domains of the bifunctional enzyme are not acting independent from each other, but are kinetically linked. Taken together, this study could elucidate the functional and structural basis of Psi-factor biosynthesis in ascomycetes and highlight the molecular background for the physiological role of Psi-factors in host-pathogen interactions, which was established by previous studies (Section 1.2.2).
Keywords: oxylipin; Double electron-electron resonance; Kinetic isotope effect; cytochrome P450; homology modeling; peroxidases; fatty acid oxidation
