Angewandte
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Chemie
The variant with all three mutations was most active, with an
state distributions of intermediates upon addition of l-serine
8-fold increase in kcat (Table 2, entry 3). The T292S mutation
by itself was also substantially activating, producing a 4-fold
increase in kcat (Table 2, entry 4).
revealed that the double mutants of PfTrpB and EcTrpB still
accumulated E(Aex1) rather than E(A-A) (Figure S7a,d).
However, the homologous double mutants of the A. fulgidus
and T. maritima enzymes showed shifted spectra in which
E(A-A) predominated (Figure S7b,c). These data suggest
that the double mutation also activates the enzymes through
allosteric mimicry, but that it does not quite reach the
activation generated by adding TrpA. This situation is
reminiscent of the earlier evolution of PfTrp0B2, wherein the
lone T292S mutation was insufficient to completely shift the
UV/Vis spectrum to E(A-A) without at least four additional
mutations.[3]
As with AfTrpS and PfTrpS, the UV/Vis absorption
spectrum of TmTrpS exhibits a strong lmax at 350 nm under
the reaction conditions. Once again, TmTrpB lacks this
probative peak and instead exhibits the lmax at 428 nm that
was observed for AfTrpB and PfTrpB. While all permutations
of the three mutations (P19G, I69V, and T292S) led to
improved kcat values (Figure S3), only the variants with T292S
exhibited a lmax at 350 nm, like TmTrpS (Figure S4). The
importance of the T292S mutation was also observed in
PfTrpB, where this mutation alone restored the kcat of the
isolated PfTrpB to that of the PfTrpS complex.[3] The effects
of this conservative mutation were even more dramatic in
TmTrpB, producing a kcat almost 3-fold higher than that of
TmTrpS (Table 2, cf. entries 4 and 5).
In the pantheon of tryptophan-derived natural products,
one can find substitution at every position on the indole
moiety (Figure 3). Position 5, for example, is chlorinated by
Mutational activation of the most distant homologue,
EcTrpB (57% identity), proved more challenging. The crucial
Thr!Ser mutation was not possible for EcTrpB because
EcTrpB already has Ser at this position (S297). Site-saturation
mutagenesis confirmed that serine is the optimal residue at
that position (Figure S5a). Unlike TmTrpB, recombination of
the 0B2 mutations in EcTrpB yielded no variants with
enhanced activity and only a few with activity similar to the
wild-type enzyme (Figure S5b).
Figure 3. Numbering of the positions on the indole moiety of trypto-
phan, and examples of natural products bearing substitutions at
position 5.
The initial screening effort with PfTrpB[3] had also
identified a variant with the mutations M144T and N166D
that was almost as active as PfTrpBT292S (Table 3, entry 1).
the halogenase PyrH en route to pyrroindomycin B (6),[4a]
and mono-oxygenated by tryptophan hydroxylase in the
biosynthesis of serotonin (7) and melatonin (8).[4d] Such
substituents have a profound effect on biological activity
because they can mask sites of metabolic degradation and
change the electronic properties of the compound. This, in
turn, alters properties like solubility and creates new binding
interactions through effects such as p stacking and halogen
bonding.[1b,13] Halogens can also provide handles for further
diversification of biologically active compounds through
cross-coupling reactions.[14] While chemical and biosynthetic
routes to tryptophan derivatives tend to be inefficient, TrpS
can provide direct access to many of these products.
Previously, however, 5-substituted indoles bearing any sub-
stituents larger than fluorine caused a substantial decrease in
activity.[1d,f,15] Furthermore, TrpS activity with electron-defi-
cient indoles has not been explored.
Table 3: Kinetic parameters of Pyrococcus furiosus TrpBM144T N166D and its
homologues.[a]
Entry
Enzyme
kcat
KM
kcat/KM
[sꢀ1
]
[mm indole]
[mmꢀ1 sꢀ1indole]
1
PfTrpBM144T N166D
EcTrpBM149T N171D
TmTrpBM145T N167D
AfTrpBM156T N178D
0.83
0.34
3.3
42
18
32
11
20
19
100
31
2[b]
3
4
0.34
[a] Assays conducted in potassium phosphate buffer (pH 8) at 758C for
Pf and Tm, 608C for Af, and 378C for EcTrpB. See Section 5.4 in the
Supporting Information for experimental details. Standard errors are
shown in Table S1. [b] EcTrpB has the following characteristics:
k
cat =0.16 sꢀ1; KM =19 mm (indole); kcat/KM =8 mmꢀ1 sꢀ1 (indole).
These two residues, unlike T292, reside in the so-called
communication (COMM)[2a] domain, which interfaces with
TrpA and undergoes large conformational motions during the
catalytic cycle. These residues are identical in the four
homologues studied here and are almost universally con-
served across all TrpBs (Figure S6). We hypothesized that the
effects of mutations at these sites might also be transferrable.
Upon making the equivalent mutations in EcTrpB, TmTrpB,
and AfTrpB, we observed activation in all variants, with
approximately 2- to 5-fold increases in kcat (Table 3, entries 2–
4).
One enzyme in our repertoire, TmTrpBM145T N167D, showed
higher activity with 5-bromoindole than even our most
optimized catalyst, PfTrpB0B2 (Figure S8). To assess whether
this was a general property of the catalyst, we compared the
relative rates of TmTrpBM145T N167D and PfTrpB0B2 with a set of
challenging 5-substituted indoles (Table 4). We then applied
N167D
TmTrpBM145T
in reactions that were run to higher
conversion in order to isolate and characterize the products.
With 5-chloroindole, the Tm variant exhibits a 3-fold rate
enhancement compared to PfTrpB0B2 (Table 4, entry 1).
Despite this improvement, the reaction still appeared to
stall at about 85% conversion when the substrates were used
in equal amounts, possibly due to competing decomposition
We wished to verify that the proteins were still being
activated by allosteric mimicry. UV/Vis analysis of the steady-
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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