MATERIALS AND METHODS Protein expression and purification

The proteins have been expressed and purified as described in (12). Mutagenesis to derive the Y183F variant of XenA has been described in (8).

Pre-steady state kinetic experiments

The oxidative half-reactions of reduced wt-XenA with 2-cyclohexenone, coumarin, 7-hydroxycoumarin and 8-7-hydroxycoumarin were measured in 50 mM Tris buffer (pH 8.0) under anaerobic conditions at 20°C as described previously (8). 70 µM of enzyme was mixed with 1600 !M of the oxidative substrate. To achieve complete reduction the enzyme it was reduced by titration with appropriate amounts of NADH in a glass tonometer with cuvette side arm. The reactions were monitored at the absorbance maximum of the FMN at 464 nm with using an Applied Photophysics SX-20MV spectrophotometer with a 1-cm observation path length cuvette. The measurements were repeated at least five times for each substrate concentration.

Crystallization and structure determination

Reduced crystals of XenA-Y183F were grown as described for the reduced wild type XenA under anoxic conditions in a glovebox (Coy type B) (8). The crystals were cross-linked for 1 hour in a harvesting solution containing 100 mM HEPES buffer (pH 7.5), 2.1 M ammonium sulfate, 4 mM NADH and 0.002% glutaraldehyde. To soak crystals with substrate, they were further incubated for 10 sec in harvesting buffer containing approximately 5 mM of the oxidative substrate. Crystals were shock frozen in harvesting buffer containing 20% (v/v) 2R,3R-butandiol and stored in liquid nitrogen.

X-ray diffraction data were collected at the beam line BL14.2 (BESSY, Berlin, Germany), at a wavelength of 0.91841 Å and 100 K. The diffraction data were processed and scaled using the XDS package (14). The structures of the XenA-Y183F variants were solved using Patterson search techniques using PHASER (15) with XenAox (PDB-Id: 2H8X (4)) as homologous search model. Subsequent rounds of model building and refinement were performed using the programs COOT (16), PHENIX (17) and Refmac5 (18).

ACKNOWLEDGEMENTS

HD was financed by the Heisenberg program of the Deutsche Forschungsgemeinschaft (DFG, DO-785/3), which also funded the project (DFG, DO-785/2).

REFERENCES

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12 Acknowledgement

First, I would like to thank Prof. Dr. Holger Dobbek for his supervision, advice and guidance - and for letting me stay part of his Bio-IN-Organic group, even though my protein did not have an iron-sulfur cluster.

I would also like to thank Prof. Dr. Matthias Ullmann and Frank Dickert for calculations on and discussions about XenA.

I am thankful to Prof. Dr. Russ Hille, who gave me the chance to work in his lab, who provided great help with the evaluation of my stopped flow data and who introduced me to Honey and Thursby. I also would like to gratefully thank Dimitri Niks, who made the stays in Riverside bodacious and for showing me all the little tricks with the stopped flow, most notably how to fix it although it is not broken.

Above all I am thankful for the great working atmosphere within our little, expanding group – Dr. Jae-Hun Jeoung, Dr. Berta M. Martins, Sandra Hennig, Sebastian Götzl, Tzong-Yuan Lin, Sophia Mende, Simon Fillenberg, Lisa Schilder, Christian-Benedikt Gerhold, Marlene Grünwald, Claudia Haas and Till Giese – thank you for helpful discussions, having a lot of fun and all the amusing Kubb games.

Moreover, many, many thanks go to Stefan Knauer for giving me crystallography lessons in every free minute and explaining over and over again, for being a friend and mostly for the encouragement on bad days.

Furthermore, I am so very thankful to my close friends Sebastian Kunze and Sebastian Wallaschek, whose support was exhaustless and who shared so many beautiful moments with me in Bayreuth or wherever we met.

I owe my deepest gratitude to my parents and wonderful friends – Petra and Uwe. Thank you so much for your guidance as well as giving me enough space to find my own way and for loving me.

Finally, my loving thanks go to Maximilian – you are my source of strength. Words fail me to express my appreciation to you. Thanks for never stop believing in me and especially for making me finish this thing!

13 Erklärung

Hiermit erkläre ich, dass ich diese Arbeit selbstständig verfasst und keine anderen als die von mir angegebenen Quellen und Hilfsmittel benutzt habe.

Ferner erkläre ich, dass ich anderweitig mit oder ohne Erfolg nicht versucht habe, diese Dissertation einzureichen. Ich habe keine gleichartige Doktorprüfung an einer anderen Hochschule endgültig nicht bestanden.

Bayreuth, den 16. August 2010