Helmholtz-Gemeinschaft

Conclusion & Outlook
two of the three analyzed aldehydes (BA t 1/2 : ca. 2.5-2.8 h; DMBA t 1/2 : ca. 4.7-5 h; 4-ClBA
t 1/2 : ca. 0.4-0.5 h). Which means that at least towards benzaldehyde and 3,5-dimethoxybenzaldehyde
a 5-10-fold stabilisation was found compared to the wildtype enzyme, while no
significant stabilisation towards 4-chlorobenzaldehyde was detected. In these BAL-variants
13 amino acids of the C-terminus were deleted, resulting in an enlarged entrance to the active
centre. For one of the variants the C-terminal His-Tag was removed and therefore a His-Tag
was fused to the N-terminus (HisBALΔ), while the C-terminal His-Tag was kept for the other
variant (BALΔHis). The results demonstrate that the C-Terminus is involved in the
inactivation process caused by the aromatic aldehydes, in addition to the previously proven
importance of the C-terminus for the catalytic activity. Furthermore, Schiff base formation of
the N-terminus with the aldehydes can be excluded, because both variants (with and without
N-terminal His-Tag) showed the same behaviour. One reason for the inactivation may be the
entrapment of the aromatic substrates and/or products in the active centre caused by the
C-terminus, leading to a local increase of reactants. This could induce hydrophobic
interactions with catalytic important residues in the active centre or the C-terminus itself.
Finally these hydrophobic interactions could lead to reversible or irreversible conformational
changes resulting in the irreversible inactivation of the BAL. The previously described
differences in the inactivation as well as in the reactivation behaviour suggest that at least two
inactivation processes are involved, which dependent on pH and the aldehyde. This is now
proven by the still relatively fast inactivation caused by benzaldehyde and 3,5-dimethoxybenzaldehyde
and the still very fast inactivation by 4-chlorobenzaldehyde.
In this work hints to the molecular reasons of the inactivation of BAL by aromatic aldehydes
were found for the first time. Whereas the previously assumed Schiff base formation with
lysine residues could be excluded as the main inactivation mechanism of BAL, opening the
excess to the active site by removing parts of the C-Terminus was at least partially successful.
Due to their 3-5-times lower activity the deletion variants are probably no alternative to the
wildtype enzyme in biotransformations. However, studies about activity, stability and micro
reaction constants of the deletion variants could provide important information about the
relevance of the C-terminus for the catalytic cycle and the structural stability of BAL. The
kinetic analysis of different conversion curves is currently done in the group of Prof. Antje
Spieß at the RWTH Aachen University. They could provide more detailed information,
whether the C-terminus really hampers the product release by shielding the active site toward
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