Mycobacterium tuberculosis H37Rv3377c encodes the diterpene cyclase for producing the halimane skeleton

Article abstract of DOI:10.1039/b415346d

The cloning and functional expression of Mycobacterium tuberculosis Rv3377c in Escherichia coli revealed that this gene encodes the diterpene cyclase for producing (+)-5(6),13-halimadiene-15-ol, which accepts geranylgeranyldiphosphate as the intrinsic substrate. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b415346d

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Mycobacterium tuberculosis H37Rv3377c encodes the diterpene cyclase
for producing the halimane skeleton{
Chiaki Nakano,a Tomoo Okamura,a Tsutomu Sato,a Tohru Dairib and Tsutomu Hoshino*a
Received (in Cambridge, UK) 4th October 2004, Accepted 15th November 2004
First published as an Advance Article on the web 4th January 2005
DOI: 10.1039/b415346d
recombinant E. coli having the chaperon-coexpression system. GC
analyses of the hexane extract from the reaction mixtures showed
that no conversion was found for GPP, FPP, squalene and
(3R,S)-oxidosqualene, but 1 was converted in a high yield (ca.
50%), indicating that Rv3377c encodes a diterpene synthase.
To isolate a sufficient amount of the enzymic product 2 for the
structure determination, 8 mg of 1 were incubated with the cell-free
homogenates from 5-L culture. The GC trace showed the presence
of both geranylgeraniol (GGOH) and 2 in addition to Triton
X-100 (Fig. 1A). The hexane extract from the incubation mixture
was subjected to SiO2 column chromatography eluting with
hexane–EtOAc (100 : 2), yielding a pure product 2 (3 mg). The
structure was determined to be 5(6),13-halimadiene-15-ol by the
detailed 2D NMR analyses (Scheme 1). The relative stereochemi-
stry of 2 was established by the NOESY spectrum. We propose to
name 2 tuberculosinol. Product 2 had a primary alcoholic group
(dH 4.10, 2H, d, J 6.5, H-15; dC 59.4, t, C-15) and no signal in the
31P NMR spectrum, suggesting that the diphosphate group of the
enzymic product was eliminated during the enzymic reaction.
Addition of phosphatase inhibitor8 led to no recovery of 2 from
the hexane extract, indicating that the polar diphosphoryl group of
the enzymic product had been eliminated by action of the
endogenous phosphatase of E. coli. In addition, from the
incubation mixture of 1 with the cell-free extract, which was
prepared from E. coli lacking the Rv3377c gene, GGOH was also
found in the hexane extract (Fig. 1B), but addition of the
phosphatase inhibitor8 led to no recovery of GGOH. These
findings indicated that Rv3377c enzyme itself had no phosphatase
activity and catalyzed only the reaction of 1 A 4. The cyclization
of 1 proceeded in a pre chair–chair conformation to give bicyclic 3
having a C8-cation. A series of 1,2-shift reactions of hydride and
The cloning and functional expression of Mycobacterium
tuberculosis Rv3377c in Escherichia coli revealed that this gene
encodes the diterpene cyclase for producing (+)-5(6),13-halima-
diene-15-ol, which accepts geranylgeranyldiphosphate as the
intrinsic substrate.
Despite over a century of research, Mycobacterium tuberculosis
(MT) remains a leading cause of infectious death worldwide. Since
the genome project finished in 1998,1 numerous studies have
appeared to elucidate the biochemical reaction of each open
reading frame. For example, sterol 14a-demethylase P450 is
demonstrated to be involved in MT H37Rv,2 suggesting the
possibility of cholesterol biosynthesis by MT. Supportive evidence
for the ability of mycobacteria to synthesize cholesterol has been
given; a trace amount of cholesterol was recently reported to be
found in M. smegmatis.3 MT H37Rv3377c protein, consisting of
501 amino acid residues,1,4 has two characteristic alignments,
called QW5 and DXDD motifs,5 which are also conserved in
oxidosqualene and squalene cyclases and/or other terpenoid
cyclases.5 To identify the biochemical function, the Rv3377c was
cloned, functionally expressed in E. coli, and the expressed protein
was subjected to the enzymic reactions using various substrates of
terpenoid cyclases. We present here the definitive evidence that
Rv3377c gene product encodes the diterpene cyclase to produce
the halimane skeleton. It should be noted that this gene is involved
only in the pathogenic species of mycobacteria.
The Rv3377c gene was amplified by PCR and cloned into the
BamH I/Hind III site of pET-22b(+). The recombinant enzyme
was expressed in E. coli BL21(DE3) under the following
conditions: isopropylthiogalactoside, 0.4 mM; induction for 10 h
at 20 uC, but almost all of the expressed protein formed an
inclusion body. A coproduction of the Rv3377c and GroE
chaperon6 in E. coli was only slightly effective for solubilizing the
recombinant protein (at most ca. 5%), but highly effective for the
enzyme activity due to the correct folding of the Rv3377c enzyme;
no enzymic activity was found unless the chaperon was
coexpressed. The following materials were tested as the substrates:
geranyl- (GPP), farnesyldiphosphate (FPP), geranylgeranyldiphos-
phate (GGPP, 1), squalene and (3R,S)-2,3-oxidosqualene. These
substrates were incubated at 25 uC and pH 7.57 for 20 h with the
cell-free homogenates containing Triton X-100 (1%), dithiothreitol
(1 mM) and MgCl2 (10 mM), which were prepared from the
{ Electronic supplementary information (ESI) available: cloning and
expression protocols of Rv3377c, amino acid alignment of some diterpene
cyclases, primers for site-directed mutants, and the enzyme purification
method. See http://www.rsc.org/suppdata/cc/b4/b415346d/
*hoshitsu@agr.niigata-u.ac.jp
Fig. 1 GC traces of the hexane extracts obtained by incubating 1 with
the cell-free extracts from E. coli having Rv3377c (A) and lacking this gene
(B). The GroE-coexpression system was used. The symbols * and # show
the Triton X-100 and impurities, respectively.
1016 | Chem. Commun., 2005, 1016–1018
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eukaryotes, as suggested for sterol 14a-demethylase and the related
sterol-metabolizing enzymes found in MT.15 Insight into the
biological function(s) of this halimane product is of particular
interest, especially from the viewpoint of the pathogenicity of MT.
The same gene as Rv3377c is also found in other pathogenic
species (M. tuberculosis CDC155116 and M. bovis subsp. bovis
AF2122/9716,17), but is not found in non-pathogens (M. smegmatis
MC2155,18 M. avium104,18 and M. avium subsp. oaratuberculosis
str.k1016). This fact strongly suggests that Rv3377c may closely
relate to the pathogenicity.19{
We thank Professor Ishii, University of Tsukuba, Japan, who
kindly provided the plasmid pT-groE (GroESL expression
vector).6 This work was made possible by the financial support
(Nos. 15380081, 15780083 and 16208012) provided by the
Ministry of Education, Science and Culture of Japan.
Scheme 1 Proposed cyclization mechanism of 1 into 4 by Rv3377c
cyclase. The diphosphoryl group was hydrolyzed by endogenous
phosphatase of E. coli. The depicted structure of 2 shows the relative
stereochemistry.
Chiaki Nakano,a Tomoo Okamura,a Tsutomu Sato,a Tohru Dairib and
Tsutomu Hoshino*a
aDepartment of Applied Biological Chemistry, Faculty of Agriculture,
and Graduate School of Science and Technology, Niigata University,
Ikarashi, Niigata, 950-2181, Japan.
E-mail: hoshitsu@agr.niigata-u.ac.jp; Fax: +81-25-262-6854
bBiotechnology Research Center, Toyama Prefectural University,
Toyama, 939-0398, Japan
methyl group in antiparallel fashion, followed by deprotonation of
H-6, introduced the double bond at C5–C6 to give 4 (Scheme 1).
Two characteristic motifs of DXDD and DDXXD are usually
found in diterpene cyclases. It is generally agreed that the DXDD
motif affords the acidic proton on the terminal double bond of
linear substrates to initiate cyclization reactions, leading to the
production of cyclic diterpenes.5,9,10 Comparison of amino acid
alignment10 between Rv3377c and other diterpene cyclases, e.g.
abietadiene and copalyldiphosphate synthases, strongly suggests
that the DXDT293–296 sequence of Rv3377c corresponds to the
DXDD motif.11 As anticipated, point mutants of D293AN and
D295AN gave no cyclization product, because of a decrease in the
acidity of the motif, but that of T296AD also unexpectedly
resulted in the loss of the cyclase activity, despite the acidity of the
DXDT motif having been increased. On the other hand, the
DDXXD motif11 is known to have a crucial role in the cyclization
reactions initiated by the release of the diphosphate group.11 This
motif is missing in Rv3377c,10 thus, 1 could be cyclized according
to Scheme 1 (protonation-initiated cyclization). A divalent cation
of Mg2+ was demonstrated to be essential to the cyclization
reaction; the homogeneous enzyme, which was purified in the
presence of EDTA, had no cyclization activity, but supplementa-
tion of MgCl2 (1 mM) afforded 2 in a high yield (94.8%).12 The
diphosphate moiety of 1 may bind to the cyclase through the
chelation of Mg2+ cation.9,11 The essentiality of the diphosphate
group for the cyclization reaction was further supported by the fact
that GGOH itself was not cyclized. Further studies on the cyclase
are underway.
Notes and references
{ Product 2. NMR data in C6D6: dH 0.81 (3H, s, H-20), 0.92 (3H, d, J 6.8,
H-17), 1.21 (m, H-1), 1.22 (3H, s, H-19), 1.27 (3H, s, H-18), 1.38 (m, H-3),
1.50 (m, H-11), 1.52 (m, H-3), 1.63 (3H, s, H-16), 1.64 (m, H-8), 1.66 (m,
H-11), 1.67 (2H, m, H-2), 1.86 (m, H-1), 1.94 (2H, m, H-7), 1.99 (ddd, J 4,
12.8, 13.2, H-12), 2.06 (ddd, J 4, 12.8, 13.2, H-12), 2.37 (bd, J 11.8, H-10),
4.10 (2H, d, J 6.5, H-15), 5.54 (bt, J 6.7, H-14), 5.67 (m, H-6); dC 15.28 (C-
17), 16.39 (C-16), 16.43 (C-20), 22.60 (C-2), 27.22 (C-1), 29.15 (C-19), 30.04
(C-18), 31.97 (C-7), 33.05 (C-12), 33.70 (C-8), 35.35 (C-11), 36.28 (C-4),
37.25 (C-9), 40.31 (C-10), 41.25 (C-3), 59.41 (C-15), 116.73 (C-6), 124.5 (C-
14), 139.2 (C-13), 146.2 (C-5). The assignments of H-18 and H-19, those of
C-18 and C-19, and those of C-16 and C-20 may be exchangeable. EIMS
(%) m/z 69 (31), 80 (100), 93 (37), 119 (80), 136 (55), 189 (81), 190 (73), 191
(68), 290 (11). HRMS (EI) calcd. for C20H34O, 290.2610; found 290.2639.
25
[a]D + 22.2 (c 0.3, EtOH).
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In conclusion, Rv3377c was demonstrated to encode a diterpene
cyclase to give the halimane skeleton. Production of diterpene
by prokaryotes is very rare. To date, only one example of
Kitasatospora griseola (formerly named Streptomyces griseolo-
sporeus) has been reported, which produces an antibiotic having a
clerodane diterpene skeleton, called terpentecin.13 Production of
the halimane skeleton by prokaryotes has not hitherto been
reported. A halimane skeleton is usually found in eukaryotes
such as plants and liverworts,14 suggesting that this gene
might have been acquired by horizontal gene transfer from
7 The optimum catalytic pH and temperature for 1 A 2 were 6.5–7.5 and
15–25 uC, respectively; see ESI.
8 Phosphatase inhibitors of cocktail 1 and 2 from SIGMA Co. were
tested. Addition of the cocktail 1 led to the recoveries of GGOH and
2 from the hexane extract of the incubation mixture, but that of
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cocktail 2 effectively inhibited the phosphatase activity of GGPP to
GGOH and that of 4 to 2.
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purified Rv3377c enzyme, further supporting the proposal that this
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diphosphate group of 4 to afford 2, acid phosphatase was added to the
preincubation mixture of 1 with the purified Rv3377c enzyme, followed
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defective in arrest of phagosome maturation, in which Rv3377c gene
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