Applications of Fluorescent Displacement-Based ... - Jacobs University

interest that originated during my BSc and MSc studies (Appendix 1-3). During
my PhD, I developed three different enzyme assays, out of which two were
designed using the supramolecular tandem assay strategy.
First, I have developed a substrate-selective tandem assay for ATPdependent
enzymes. This project has been a continuation of my MSc studies and
been finalized and published during my PhD time (Appendix 4). The study deals
with following ATP consumption by dephosphorylation caused by an enzyme
called potato apyrase. This particular assignment also allowed the implementation
of anion-receptor macrocycles, namely an amino-derivative of γ-cyclodextrin and
a cyclophane, into the tandem assay methodology. In addition to enzymatic
conversion monitoring, the assay has been implemented for screening of
activators and it was transferred to other nucleotides.
The second assay was designed for Dim-5 from Neurospora crassa. This
is a histone lysine methyltransferase, which specifically trimethylates K9 of the
histone H3 tail and which was provided to us as part of a collaboration with the
Jeltsch research group at Jacobs University Bremen. Assays for this enzyme were
previously restricted to the use of radioactive labels or more laborious techniques.
By means of the developed product-selective tandem assay, its activity can be
followed in a continuous and label-free fashion. Additionally, its functionality
was also illustrated by performing inhibition studies, rendering it suitable for
future screenings.
The third project focuses on the conversion of aspartic acid to β-alanine,
which is carried out by L-aspartate-α-decarboxylase and which was part of a joint
project with the National University of Singapore. The purpose of the study has
been a systematic screening for inhibitors against L-aspartate-α-decarboxylase,
which has a crucial role for the virulence and persistence of the bacterial agent
causing tuberculosis, Mycobacterium tuberculosis. Unlike the previous two
enzyme assays, the enzymatic transformation, namely the depletion of aspartic
acid and concomitant formation of β-alanine was followed using nuclear magnetic
resonance (NMR) spectroscopy. The inhibitory effect of several known as well as
novel inhibitors identified via bioinformatics could be conveniently established by
NMR studies.
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