D. Tran et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6838–6841
6841
Figure 4. Reaction of racemic (RS)-2-deoxyE4P catalysed by P. furiosus DAH7P synthase, showing initial fast rate corresponding to the use of (3S)-2-deoxyE4P (equivalent to
the rate of reaction in the presence of the enantiopure (3S)-2-deoxyE4P) followed by slow reaction rate due to consumption of the (3R)-2-deoxyE4P. The reaction progress is
followed by the loss of PEP at 232 nm as described in the Supplementary data.
for this mixture was virtually unchanged (2600 330
l
M)
article can be found, in the online version, at doi:10.1016/
– although, it should be noted that KM for (S)-2-deoxyE4P and (R)-
3-deoxyE4P are very much larger compared to E4P for the E. coli
DAH7P synthase than for the M. tuberculosis and P. furiosus DAH7P
synthases. In contrast the type II M. tuberculosis DAH7P synthase
was intolerant to variation of configuration at C3, with the racemic
3-deoxyE4P substrate utilising only 50% of the racemic mixture,
presumably the (R)-3-deoxyE4P based on the enantiopure results.
The conformational flexibility that allows both enantiomers of
2-deoxyE4P to be substrates for P. furiosus DAH7PS allows the (S)
enantiomer of 3-deoxyE4P to bind in a manner that precludes the
binding and reaction observed for enantiopure (R)-3-deoxyE4P.
In summary, both 2- and 3-deoxy variants of E4P were found to
be substrates for both types I and II DAH7P synthases. This indi-
cates that whereas the hydroxyl groups, and in particular the C3
hydroxyl group of this aldehydic substrate support efficient reac-
tion, they are not essential for DAH7P synthase catalysis. The
observation that 3-deoxyE4P acts as an alternative substrate
strongly supports an acyclic reaction mechanism for the genera-
tion of DAH7P by this enzyme.
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We gratefully acknowledge the New Zealand Marsden Fund
(UOC710) and the Massey University Research Fund for financial
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Supplementary data
Supplementary data associated (details of synthetic procedures
for the preparation of 3-deoxyE4P and the kinetic assays) with this