Wolframabhängige Aldehydoxidoreduktase - Neue Einblicke in Katalyse, Struktur und Kofaktor-Maturation
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Philipps-Universität Marburg
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Abstract
Aldehyde Oxidoreductase from Aromatoleum aromaticum belongs to the clade of bacterial
AORs and consists of three different subunits in a α2β2γ composition. It catalyzes the
oxidation of various aldehydes to the corresponding carbonic acids. The β subunit contains
a tungsten-bis-pterin cofactor (WCo) in the active site. Via a [4Fe-4S] cluster in the β
subunit and four further [4Fe-4S] clusters in the α subunit, electrons can be transferred
to a FAD molecule located in the γ subunit. Activity measurements in vitro are possible
with benzyl viologen or NAD+ as electron acceptor suggesting that either NAD+ or a
ferredoxin acts as the physiological electron acceptor. By the use of suitable reductants
like Ti(III) or Eu(II) it is also possible to observe the reduction of carbonic acids to the
aldehydes. Furthermore, AOR posesses hydrogenase activity, which allows the reduction
of benzyl viologen, NAD+ or even carbonic acids using elementary hydrogen. Besides the
three structural genes aorA-C, the aor operon contains the two additional genes aorD and
aorE that code for putative maturation factors.
This work shows the generation of an expression system using Aromatoleum evansii
KB740 as host organism that allows recombinant production and affinity chromatography
based purification of AOR. After optimization of the experimental conditions, the activity
of the recombinant AOR exceeds the activity of natively purified AOR by considerable
amounts. This work also shows determination of tungsten and iron contents as well as
FAD and furthermore contains some experiments regarding the hydrogenase activity.
Besides the enzyme itself and its potential biotechnological applications, also the WCo
and its maturation arise interest. The biosynthesis of the WCo mostly follows the wellinvestigated
pathway of molybdenum cofactor (MoCo) synthesis, nevertheless WCo gets
inserted very specifically into the AOR despite the chemical similarities between tungsten
and molybdenum. The two paralogues of molybdopterin molybdotransferase MoeA1 and
MoeA2 have been discussed in literature as a potential explanation for this discrimination
process.
To elucidate the role of these two proteins in vivo, ΔmoeA deletion mutants of A.
evansii were constructed. Using these strains, it could be shown that synthesis of the
WCo strictly relies on MoeA2 while assembly of the MoCo works with either of the two
paralogues. Taken together with the observation that the activity of AOR heavily depends
on the presence of the maturation factors AorD and/or AorE, it is possible to postulate a
mechanism for WCo maturation that explains the selective incorporation of tungsten into
the enzyme.
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Dates
Created: 2022Issued: 2023-04-05Updated: 2023-04-05
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
AOR Wolframenzym Wolfram-Kofaktor Aldehydoxidation Molybdän-Kofaktor WCo MoCoTungsten Enzyme Tungsten Cofactor Aldehyde Oxidation Molybdenum Cofactor WCo MoCo
DDC-Numbers
570
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Hege, Dominik Alexander (0000-0002-2179-8037): Wolframabhängige Aldehydoxidoreduktase - Neue Einblicke in Katalyse, Struktur und Kofaktor-Maturation. : Philipps-Universität Marburg 2023-04-05. DOI: https://doi.org/10.17192/z2023.0222.