Dissertation / PhD Thesis

PUBDB-2025-04019

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Biophysikalische Untersuchung von an Biotransformationbeteiligten Flavinmonooxygenasen

kraus, y. (Corresponding author)

2025

Abstract: This work deals with the expression, purification, characterization, and crystallization of ΔhFMO5,a variant of the human flavin-dependent monooxygenase 5 (hFMO5) without transmembraneregion. This modification enables the expression and purification of soluble protein withoutaddition of detergent, resulting in the isolation of active protein saturated with FAD. Theaggregation prone ΔhFMO5 was successfully expressed in a fermenter using the strain E. coliRosetta 2 (DE3) pLysS, lysed by enzymatic cell disruption and purified through affinity and sizeexclusion chromatography. Characterization of ΔhFMO5 via mass photometry reveals that insolution there is present a mixture of different oligomeric states, with the tetrameric form beingpredominant. The catalytic cycle of ΔhFMO5 includes an uncoupled metabolic pathway, in whichNADPH is consumed to regenerate the FAD cofactor without substrate turnover, leading to theformation of hydrogen peroxide. Iterative modeling of the reaction kinetics of ΔhFMO5demonstrates that substrate metabolism as a second-order reaction proceeds more slowlycompared to the uncoupled reaction. Activity measurements show that the conversion ofphenylacetone to benzyl acetate is catalyzed by ΔhFMO5 with a turnover rate of 5.1 %. N-acetylserotonin was identified as a potential endogenous substrate playing a role in the productionof melatonin and serotonin.

Note: Dissertation, Christian - Albrechts - Universität zu Kiel, 2025

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Contributing Institute(s):

1. EMBL-User (EMBL-User)

Research Program(s):

1. 6G3 - PETRA III (DESY) (POF4-6G3) (POF4-6G3)

Experiment(s):

1. PETRA Beamline P12 (PETRA III)

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