Communications
different from that in 1 (C-14; Scheme 2C and D). We propose
that Y167 may interact with the methyl residue at C-15 of 3 to
prevent cyclization of the C ring (Scheme 2C) and may not in-
teract with the methyl residue at C-14 of 1 (Scheme 2D). The
pre-C ring of 3 to form 10 is boat form (Scheme 2B), which
suggests that A167 of TCY167A interacts with the methyl residue
at C-15 to prevent the formation of the stable chair form in
cyclization of the C ring. This also supports our hypothesis.
As W169 is located at the opposite side of the methyl resi-
due of C-15 of 3 in SHC (Scheme 2A and Figure S2), it is not
expected to prevent the cyclization of 3 at the bicyclic step. In
the modeled structure, Y167 is positioned near W169 and at
the opposite side toward the methyl residue of C-15 (Fig-
ure S2). However, the present study proposes that the actual
position of Y167 may be near the methyl residue of C-15.
TCY167W, which is similar to wild-type SHC, stopped cyclization
at the bicyclic step in the first reaction (Table 1), which sup-
ports the hypothesis that the location of Y167 in TC is different
from the location of W169 in SHC in the active-site cavity.
Whereas active-site residues of SHC are highly conserved in
TC, F601 of SHC is not conserved and corresponds to L596 in
TC (Scheme 2A, Figures S1 and S2), which is a characteristic of
TC according to Bosak et al. and our previous work.[4,9] Given
that F601 may function to stabilize the secondary carbocation
(C-14 and C-18) of tricyclic and tetracyclic intermediates
through cation–p interactions in SHC (Scheme 2A),[6] it is possi-
ble that the lack of p electrons at position 596 might stop cyc-
lization at the tricyclic step in TCY167A. According to a previous
Experimental Section
General procedure and materials: NMR spectra were recorded by
using a Bruker DPX 400 spectrometer at 400 MHz (1H) and
100 MHz (13C). GC–MS was performed with a JMS-T100GCV spec-
trometer (Jeol, Tokyo, Japan) equipped with a DB-1 capillary
column (30 mꢁ0.25 mmꢁ0.25 mm; J&W Scientific, Inc., Folsom, CA,
USA) in the EI mode operated at 70 eV. HRMS was performed by
using a JMS-T100LP spectrometer (JEOL) in ESI mode. GC analyses
were performed by using a Shimadzu GC-2014 chromatograph
equipped with a flame-ionization detector and a DB-1 capillary
column (30 mꢁ0.25 mmꢁ0.25 mm; J&W Scientific, Inc.). Compound
1 was previously isolated from B. subtilis.[5a] Compound 3 was pur-
chased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Site-directed mutagenesis: The QuikChange Site-directed Muta-
genesis Kit (Stratagene) was used to introduce the desired muta-
tions (Y167A) by following the manufacturer’s instructions by
using pairs of mutagenic primers (Y167A fwd: 5’-GAATT AAGCA
CTGCT GCCAG AATTC ACTTC GTTCC GATG-3’, Y167A rev: 5’-CATCG
GAACG AAGTG AATTC TGGCA GCAGT GCTTA ATTC-3’). Y167F,
Y167L, Y167W, and Y167A/L596F were synthesized by GENEWIZ,
Inc. (Suzhou, China).
Enzymatic assay for wild-type and mutant TCs: Escherichia coli
BL21(DE3) harboring pColdTF-BmeTC[1] was grown at 378C in ly-
sogeny broth (LB; 1 L) with ampicillin (100 mgmLÀ1). Expression of
the recombinant protein was induced by adding isopropyl-b-d-thi-
ogalactopyranoside (IPTG, 0.1 mm) once the OD600 reached approx-
imately 0.6. Further cultivation of BL21(DE3) recombinants was per-
formed for 12 h at 158C. E. coli cells expressing recombinant TC
were harvested by centrifugation and were resuspended in buf-
fer A (30 mL) containing Tris·HCl (50 mm, pH 7.5), dithiothreitol
(2.5 mm), EDTA (1 mm), and 0.1% Triton X-100. The cells were dis-
rupted by sonication with UP200s (Hielscher Ultrasonics GmbH,
Teltow, Germany) at 4–108C for 15 min. The resulting suspension
was centrifuged at 10000g for 20 min. The pellet was discarded,
and the resulting supernatant was used as a cell-free extract. The
mixture for analyzing the enzymatic activity of TC contained sub-
strate (1: 0.05 mg and 3: 0.1 mg) emulsified with Triton X-100
(2 mg) in buffer A (1 mL) and cell-free extract (4 mL) containing TC
in a total volume of 5 mL. The reaction was performed at 308C for
64 h and was terminated by using a 15% KOH/MeOH solution
(6 mL). The lipophilic enzymatic product was extracted from the in-
cubation mixtures with n-hexane (3ꢁ5 mL). Triton X-100 detergent
was removed by passing the extract through a short SiO2 column
(n-hexane/EtOAc=100:20) and then subjecting the eluent to GC
and GC–MS. GC conditions for the products from substrate 3 were
as follows: injection temperature=3008C, column temperature=
220–2808C (18CminÀ1), flow rate (He gas)=1.30 mLminÀ1. GC
conditions for the products from substrate 1 were as follows: in-
jection temperature = 3008C, column temperature = 220–3008C
(38CminÀ1), flow rate (He gas) = 1.30 mLminÀ1. Enzyme assays for
mutated TCs were performed following the same methods as
those used for TC.
study, SHCF601A produces tricyclic products,[10] similar to TCY167A
.
Thus, we prepared TCY167A/L596F, which is similar to wild-type
SHC, and analyzed products formed by incubation with 3.
However, no new product possessing a tetra- or pentacyclic
skeleton, except for 10, was detected (Table 1), which indicated
that F596 could not stabilize the secondary carbocation (C-14)
in the mutant. Whereas the position of L596 was almost identi-
cal to that of F601 in the modeled structure of TC (Figure S2),
the actual location of L596 may be different from that in the
model.
Recently, new onoceroid synthases were identified in
ferns.[11,12] Ferns utilize two enzymes for onoceroid biosynthe-
sis.[11,12] One fern onoceroid synthase, pre-a-onocerin synthase
(LCC), catalyzes the formation of a bicyclic compound. Araki
et al. pointed out that its sequence (254MNIHG258) was notably
different from that of lanosterol synthase. H257 of LCC corre-
sponded to Y167 of TC (Figure S4). Although Araki et al. specu-
lated that the sequence was responsible for recognizing the
terminal epoxide of the substrate,[11] H257 of LCC might also
function to terminate cyclization at the bicyclic step, similar to
Y167 of TC.
Isolation of product 10 synthesized from 3 by TCY167A: The cell-
free extracts (720 mL) were prepared in a manner similar to that
described above from E. coli BL21(DE3) harboring pColdTF-TCY167A
cultured in LB (24 L). To isolate product 10 formed by TCY167A, com-
pound 3 (45 mg) was emulsified with Triton X-100 (900 mg) in buf-
fer A containing Tris·HCl (50 mm, pH 7.5), dithiothreitol (2.5 mm),
EDTA (1 mm), and 0.1% Triton X-100 and was then incubated with
the cell-free extracts (720 mL) at 308C for 64 h. After 15% KOH/
MeOH solution (1.08 L) was added to the mixture, the lipophilic
In conclusion, this is the first analysis of the catalytic mecha-
nism of an onoceroid synthase that makes use of mutated en-
zymes. The bulk size at the Y167 position was found to be im-
portant for termination of the cyclization of 3 at the bicyclic
step. The active-site structure of TC would be slightly different
from that of SHC. Analysis of the 3D structure of TC is necessa-
ry to elucidate the catalytic mechanism of TC and will facilitate
the efficient biosynthesis of bioactive onoceroids, such as 8.
ChemBioChem 2017, 18, 1 – 5
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ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ
Tenkovskaia, Liudmila
