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resulting in monocyclic products. Furthermore, steric inter-
action of the C1-methyl group of the substrate 1c and the
leucine at position 607 can be assumed in this pre-folding state
(Figure 2B, dots). Interestingly, the G600T-hydroxy group
would be too far away for hydrogen-bonding in this state
(Figure 2B, pre-folding state 2), which makes its significance
in the catalysis hard to pin down (cf. Mechanistic studies).
Based on the docking results, we performed site-directed
mutagenesis at the position L607 and determined amino
acids—smaller in size than leucine—as beneficial for the
monocyclization reaction (Figure S7B). Remarkably, a com-
bination of variant II with the best mutation L607S did not
lead to the best performing double-variant, which we assume
is due to earlier reported antagonistic epistatic effects
(Table S10).[36] Instead, variant III (G600T/L607A) turned
out to be the best double variant showing almost twice the
total turnover number (114 ꢁ 13) compared to II (62 ꢁ 2) and
higher selectivity (79%) towards the desired g-dihydroionone
3 (II: 50%) (Figure 2C,D, III). Building on the docking
results, we disrupted the hydrogen-bonding at position Y420
with the carbonyl moiety of the substrate 1c to prevent pre-
folding state 1 and thereby bicyclization (Figure 2C, D, IV).
The resulting variant IV showed 260 ꢁ 4 total turnover with
a selectivity of 94% towards the desired product 3. Bicycliza-
tion was reduced to only 2% and the traces of the hydration
product were completely eliminated (cf. supporting chroma-
togram G11). Finally, one further round of site-saturation
mutagenesis by employing the 22c-trick[37] at sites adjacent to
position 600 and 607 resulted in variant V with a slightly
increased steric bulk at position 306 (A!V). The 22c-trick is
used in directed evolution of enzymes to reduce the screening
effort of protein libraries (for details see ref. [36]). The total
turnover number was increased 157-fold compared to the
native enzyme and selectivity towards the desired product 3
(Figure 2C, D, V) was at 97%. Finally, we performed sulfuric
acid-catalyzed cyclization of g-dihydroionone 3 to obtain
enantiopure (+)-a-ambrinol 7 in only two (bio-)synthetic
steps compared to the five chemosynthetic steps for enan-
tioenriched material.[26] Please note that as of now there is no
chemical catalyst known which is able to direct the cascade
progress (99:1 monocyclization vs. bicyclization) with high
product selectivity (97% g) and excellent enantiocontrol
(> 99.5% ee) (Figure 2E and Figure S11).
Scheme 1. Biotransformation of neryl acetone 1c and geranyl acetone
1t with variant G600R. The E-Isomer 1t was almost fully converted to
the bicyclic product 2t (red, bottom) and the Z-isomer 1c to bicyclic
product 2c (red, top) as well as monocyclic products 3 (blue) and 4
(grey). Relative conversions are given below the products (for reaction
conditions see table S7).
the cascade towards monocyclization, as well as to control this
reaction in terms of product- and stereoselectivity. As starting
point for the engineering, we decided to pay particular
attention to position G600 and saturated this position in order
to examine each amino acid in the cyclization reaction. The
results disclose that mainly small and polar amino acids drive
the monocyclization reaction at position G600. In terms of
selectivity, G600T (Figure S7A), that is, variant II, performed
best (Figure 2C, D, II). Product 5 was not observed at all and
the amount of hydration product 6 was negligible (supporting
chromatogram G10). In order to rationalize these findings, we
performed docking studies of substrate 1c in the active site of
variant II with YASARA.[32] We are aware of the challenges
that arise with docking substrates and homology modelling of
terpene cyclases.[33] However, based on results that show the
substrate-bound[34] and the apo-form[19] as essentially equal
crystal structures and comparing the pre-folding of the
squalene-analog aza-squalene bound[34] (Figure S8) with our
docking results, we were confident with our approach.
Thereby, we wanted to determine the most probable pre-
folding states of neryl acetone 1c and locate mutational hot
spots.
Docking results are depicted in Figure 2B and suggest two
major pre-folding states: Pre-folding state 1 favors bicycliza-
tion due to the coordination of the carbonyl moiety by the
Y420-hydroxy group. The resulting second carbocation of the
cation cascade reaction (Figure 2B, red charge) may interact
with one lone-pair of the oxygen and thereby form a covalent
bond.[35] Our experimental results demonstrate how bulky
amino acids at position G600 favor this pre-folding state
(Figure 2C, G600R and Figure S7A). However, there is
a turning point at the size of threonine, where polarity seems
to play a more significant role (Figure S7A, G600T and
G600V). Earlier studies by Peters and co-workers on terpene
cyclases demonstrated that the introduction of threonines or
serins as catalytic Brønsted bases adjacent to an intermediary
carbocation result in deprotonation and consequently cascade
interruption.[17] However, we assume that this is due to
hydrogen-bonding capabilities of polar residues. Pre-folding
state 2 shows the carbonyl moiety hydrogen-bonded by
G600T and Y609. Thus, the lone-pairs of the oxygen are
facing away from the resulting second carbocation, ultimately
Mechanistic Studies and Substrate Scope
Encouraged by this high selectivity towards the formation
of the desired product, we wanted to shed light on the
underlying mechanism. Therefore, we performed a system-
atical deconvolution approach of the variant IV (as the
product selectivity was already very high at this point) at
positions G600 (A), L607 (B) and Y420 (C). In order to
highlight the beneficial hydrogen-bond at position Y609 (D),
we further disrupted the hydrogen-bond here (Y!F) and
combined this mutation with selected deconvolution variants.
Figure 3A shows the resulting activities and selectivities of
the generated variants compared with variant IV, whereas
a letter means a mutation at the above-mentioned position
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Angew. Chem. Int. Ed. 2021, 60, 2 – 8
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