New Diterpene Synthase from M. tuberculosis
77
tions of 3, ranging from 20 mM to 100 mM, and the kinetic parameters
were deduced by non-linear least-squares fitting to the Michaelis–
Menten equation.
3
4
5
2
1
kDa
75
GroE
Enzymatic syntheses of 2 and 5 by the MBP fusion Rv3378c enzyme.
The incubation conditions were essentially the same as those reported
by Mann et al.11) Substrate 3 (50 mg) was incubated at 30 ꢂC with the
purified protein (0.575 mg) in a solution containing 50 mM HEPES at
pH 7.5, 10% glycerol, 1 mM KCl, and 10 mM MnCl2. The GC-MS
analysis we conducted showed two peaks for 2 and 5 that appeared in a
1:1 ratio (Supplemental Fig. S11).
50
37
Rv3378c
protein
Spectroscopic data for 2, 5a, and 5b. Product 2. The detailed NMR
and MS data have been given in the previous paper4) (see also
Supplemental Fig. S5b).
25
10
Product 5a. NMR data (Supplemental Figs. S7 and S8). 1H-NMR
(600 MHz, C6D6,) ꢀ: 0.831 (Me-20, 3H, s), 0.935 (Me-17, d,
J ¼ 6:7 Hz), 1.19 (H-1, m), 1.213 (Me-19, 3H, s), 1.242 (Me-16, 3H,
s), 1.264 (Me-18, 3H, s), 1.39 (H-3, m), 1.45 (H-11, m), 1.50 (H-12,
m), 1.52 (H-3, m), 1.61 (H-8, m), 1.62 (H-12, m), 1.68 (H-2, 2H, m),
1.94 (H-1, m), 1.96 (H-7, 2H, m), 160 (H-11, m), 2.38 (H-10, bd,
J ¼ 12:5 Hz), 5.08 (Hb-15, dd, J ¼ 10:7, 1.3 Hz), 5.30 (Ha-15, dd,
J ¼ 17:2, 1.3 Hz), 5.67 (H-6, bs), 5.90 (H-14, dd, J ¼ 17:2, 10.7 Hz);
13C-NMR (150 MHz, C6D6) ꢀ: 15.28 (C-17, q), 16.48 (C-20, s), 22.55
(C-2, t), 27.63 (C-1, t), 28.22 (C-16, q), 29.24 (C-19, q), 30.06 (C-18,
q), 30.39 (C-11, t), 32.04 (C-7, t), 33.66 (C-8, d), 35.46 (C-12, t), 36.26
(C-4, s), 36.95 (C-9, s), 40.20 (C-10, d), 41.23 (C-3, t), 72.93 (C-13, s),
111.4 (C-15, t), 116.8 (C-6, d), 145.7 (C-14, d), 146.2 (C-5, s). EIMS
(%) (Supplemental Fig. S7a) m=z: 80 (75), 119 (48), 136 (23), 175
(13), 191 (100), 272 (0.92), 290 (Mþ, 0.03). HR-EIMS m=z: found,
Fig. 2. SDS–PAGE of the Rv3378c-Encoded Protein Expressed by
pET-16b Carrying the Rv3378c Gene in E. coli BL21 (DE3).
Lane 1, molecular mass marker; Lane 2, pET-16b/BL21
(DE3) + GroE (no incorporation of the Rv3378c gene); Lane 3,
pET-16b-Rv3378c/BL21 (DE3) + GroE, soluble protein; Lane 4,
pET-16b-Rv3378c/BL21 (DE3) + GroE, insoluble protein; Lane 5,
pET-16b-Rv3378c/BL21 (DE3) + GroE, purified in an Ni-NTA
column.
A
B
25
290.2603 (Mþ, C20H34O requires 290.2610). ½ꢁꢃD þ33:0 (c 0.05,
EtOH).
Product 5b. NMR data (Supplemental Figs. S9 and S10). 1H-NMR
(600 MHz, C6D6) ꢀ: 0.831 (Me-20, 3H, s), 0.959 (Me-17, d, J ¼
6:7 Hz), 1.19 (H-1, m), 1.211 (Me-19, 3H, s), 1.242 (Me-16, 3H, s),
1.265 (Me-18, 3H, s), 1.38 (H-3, m), 1.40 (H-11, m), 1.52 (H-12 and
H-3, 3H, m), 1.55 (H-11, m), 1.62 (H-8, m), 1.69 (H-2, 2H, m), 1.92
(H-1, m), 1.96 (H-7, 2H, m), 2.36 (H-10, bd, J ¼ 12:6 Hz), 5.08
(Hb-15, dd, J ¼ 10:7, 1.3 Hz), 5.30 (Ha-15, dd, J ¼ 17:3, 1.3 Hz), 5.67
(H-6, bs), 5.90 (H-14, dd, J ¼ 17:3, 10.7 Hz); 13C-NMR (150 MHz,
C6D6) ꢀ: 15.24 (C-17, q), 16.47 (C-20, s), 22.57 (C-2, t), 27.64 (C-1, t),
28.26 (C-16, q), 29.25 (C-19, q), 30.06 (C-18, q), 30.38 (C-11, t), 32.03
(C-7, t), 33.68 (C-8, d), 35.47 (C-12, t), 36.26 (C-4, s), 36.95 (C-9, s),
40.20 (C-10, d), 41.23 (C-3, t), 72.94 (C-13, s), 111.4 (C-15, t), 116.8
(C-6, d), 145.7 (C-14, d), 146.1 (C-5, s). EIMS (%) (Supplemental
Fig. S9a) m=z: 80 (78), 119 (52), 136 (21), 175 (12), 191 (100), 272
(0.77), 290 (Mþ, 0.03). HR-EIMS m=z: found, 290.2603 (Mþ,
5
2
20
30
10
10
20
30
Retention time (min)
Fig. 3. GC Traces of the Hexane Extract from the Reaction Mixture
of 3 with the Purified N-Terminal His-Tagged Rv3378c Enzyme (A)
and without the Rv3378c Enzyme (B).
Two peaks of 2 and 5 appeared as the enzymatic products (A), but
no production of 2 was apparent without this enzyme, indicating that
no hydrolysis reaction of 3 to give 2 had occurred in the absence of
this enzyme and under the work-up conditions. Similar GC
chromatograms were obtained for the C-terminal His-tagged and
MBP fusion enzymes, indicating that the ratio of 2 to 5 was
approximately 1:1 for the three expressed enzymes. The GC
conditions were as follows: capillary column, DB-1 (0:25 mm ꢁ
30 m); initial temperature, 170 ꢂC; final temperature, 270 ꢂC; rate,
3 ꢂC/min; carrier gas pressure, 0.5 kg/cm2.
25
C20H34O requires 290.2610). ½ꢁꢃD þ32:1 (c 0.05, EtOH).
Results and Discussion
Expression of the Rv3378c-encoded enzymes and the
enzymatic products
We have revealed in our previous studies4,5) that the
Rv3377c enzyme catalyzed the conversion of 1 into 3.
Naturally occurring diterpenes are usually isolated in a
PP-released form. We searched for the gene(s) homol-
ogous to terpene synthases that were involved in the
H37Rv genome, but no homologous gene other than
Rv3377c could be found by a BLAST search. We
postulated that the flanking gene, Rv3376 or Rv3378c,
may encode a dephosphorylating enzyme, because
both gene products possess the characteristic DDXXD
motif that is highly conserved in diterpene cyclases6)
(Supplemental Fig. S1). Both genes were amplified by
PCR and cloned into the NdeI and BamHI sites of the
pET16b vector. Figure 2 shows the SDS–PAGE results
for the Rv3378c protein expressed in E. coli BL21
(DE3). The expressed proteins almost formed inclusion
bodies. However, coexpression of the Rv3378c proteins
and GroE chaperones15) in E. coli was effective for
obtaining the active soluble enzyme. Purification in an
Ni-NTA affinity column afforded a single band on
SDS–PAGE (Fig. 2 and Supplemental Fig. S2). Since
the crude cell-free extract obtained from E. coli had
phosphatase activity,4,5) we completely purified the
protein to examine the enzymatic reaction. Incubating
3 with the purified N- and C-terminal His-tagged
Rv3378c enzymes resulted in two peaks of 2 and 5 in
an approximate 1:1 ratio as shown in the GC trace
(Fig. 3A). It should be noted that 2 was not available
from the mixture incubated in the absence of the
purified Rv3378c enzyme, definitively indicating that 2