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Organic & Biomolecular Chemistry
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Organic & Biomolecular Chemistry
with solvent signals), 3.14 (m, 1H), 2.13 (m, 2H), 2.07 (m, 2H), 1.76
(d, 0.8, 3H), 1.65 (d, 1.0, 3H), 1.58 (d, 0.6, 3H).
Conflicts of interest
The authors declare no competing financDiaOlIi:n1t0e.1r0e3s9t/.C8OB02037J
Determination of kinetic parameters
Kinetic parameters were determined by incubation of an
appropriate amount of purified recombinant FgaPT2 or mutant with
1 mM of 1 and varied concentrations of DMAPP or GPP as prenyl
donors at 37 °C. An optimal protein amount and incubation time
were determined by measuring protein and time dependency. The
enzyme assays were performed in duplicate. Protein concentration
of 0.1 µM for FgaPT2 and an incubation time of 2 min were applied
to determine the enzyme kinetics for DMAPP. To determine the
kinetic parameters of FgaPT2 mutants for GPP, 1.9 µM of M328X (X
= C, A, T, S, G and V) and 3.8 µM of M328N were incubated for 20
min. In a total volume of 50 µL, each reaction mixture contained
DMAPP or GPP in concentrations of up to 0.5 or 1.0 mM, 1.0 mM of
1, 10 mM CaCl2, up to 3.6% (v/v) glycerol, and 50 mM Tris-HCl (pH
7.5). The reactions were terminated with 50 µL MeOH and
subsequently centrifuged at 13,300 rpm for 20 min. A volume of 80
µL supernatant was injected and analysed by HPLC.
Acknowledgements
We thank Wolfgang Brandt (IPB, Halle) for suggestions, Lena
Ludwig-Radtke, Rixa Kraut, Stefan Newel (University of
Marburg) for synthesis of prenyl donors, taking MS and NMR
spectra, respectively.
Funding information The Bruker micrOTOF QIII mass
spectrometer was financially supported in part by a grant from
the Deutsche Forschungsgemeinschaft (INST 160/620-1 to S.-
M. L.).
Notes and references
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Molecular modelling and docking
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The conformations of the mutants were calculated in silico using
foldx.30,31 Substrate docking was done manually for GPP and FPP
using COOT32 and DMAPP of FgaPT2 (PDB code: 3I4X) as a template.
The conformation of the prenyl donors were energetically
optimized. Docking of 1 was performed by in silico docking using
vina.33 The possible docking poses were analysed and verified for
chemical sense of the observed catalytic reaction. Figures were
generated with pymol (The PyMOL Molecular Graphics System,
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Conclusions
In summary, we demonstrated a convenient approach to obtain
modified enzymes in which the substrate specificities differ clearly
from the wild-type enzyme and from each other. By structure-
based modelling of the tryptophan C4-dimethylallyl transferase
FgaPT2, we are able to increase GPP and FPP acceptance by the
mutation of gatekeeping residues. To the best of our knowledge, a
natural or unnatural geranyl transferase or farnesyltransferase for
tryptophan as a free amino acid has not been reported prior to this
study. Our results provide evidence that a switch of the prenyl
donor acceptance is not only possible for enzymes with high
flexibility for these substrates such as AtaPT, MpnD and TleC,15,16
but can also be applied for enzymes which are practically limited to
the use of DMAPP as donor. Several mutants show high activity
towards GPP and could already be used as geranyl transferases for
chemoenzymatic synthesis and synthetic biology. Our findings
should encourage the PT community to manipulate additional ones
of the approximately 45 DMATS-type PTs, which mainly use DMAPP
as prenyl donor.5 The farnesyltransferase activities are necessary to
be optimized by identification of other important factors and
additional mutations. This would require additional structural data,
e. g. of the engineered enzymes.
23 A. B. Woodside, Z. Huang, and C. D. Poulter, Org. Synth.,
1988, 66, 211-215.
8 | Org. Biomol. Chem, 2018, 00, 1-9
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