Incubation of 2-methylisoborneol synthase with the intermediate analog 2-methylneryl diphosphate

Article abstract of DOI:10.1038/ja.2017.24

Incubation of synthetic 2-methylneryl diphosphate (2-MeNPP, 10) with 2-methylisoborneol synthase (MIBS) gave a mixture of products that differed significantly from that derived from the natural substrate (E)-2-methylgeranyl diphosphate (3, 2-MeGPP). The proportion of (-)-2-methylisoborneol (1) decreased from 89 to 17% while that of 2-methylenebornane (4) increased from 10 to 26%, with the relative yields of the isomeric homo-monoterpenes 2-methyl-2-bornene (5) and 1-methylcamphene (6) remaining essentially unchanged (<1% each), as determined by chiral GC-MS analysis. The majority of the product mixture resulting from the MIBS-catalyzed cyclization of 2-MeNPP (10) consisted of the anomalous monocyclic homo-monoterpenes (±)-2-methylllimonene (15, 39%) and 2-methyl-α-terpineol (13, 10%), as well as the acylic derivatives 2-methylnerol (11, 7%) and 2-methyllinalool (14, <1%). The steady-state kinetic parameters of the MIBS-catalyzed reaction, determined using [1- 3 H]-2-methylneryl diphosphate (2-MeNPP), were k cat 0.0046±0.0003 s -1, K m 18±6 μm and k cat /K m 2.55 × 10 2 M -1 s -1. In comparison, the natural substrate 2-MeGPP had a k cat 0.105±0.007 s -1, K m 95±49 μm and k cat /K m 1.11 × 10 3 M -1 s -1. Taken together with earlier X-ray crystallographic studies of MIBS, as well as previous investigations of the mechanistically related plant monoterpene cyclase, bornyl diphosphate synthase, these results provide important insights into the binding and cyclization of both native substrates and intermediates and their analogs.

Full text of DOI:10.1038/ja.2017.24

The Journal of Antibiotics (2017), 1–7
2017 Japan Antibiotics Research Association All rights reserved 0021-8820/17
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&
ORIGINAL ARTICLE
Incubation of 2-methylisoborneol synthase with the
intermediate analog 2-methylneryl diphosphate
Wayne KW Chou, Colin A Gould and David E Cane
Incubation of synthetic 2-methylneryl diphosphate (2-MeNPP, 10) with 2-methylisoborneol synthase (MIBS) gave a mixture of
products that differed significantly from that derived from the natural substrate (E)-2-methylgeranyl diphosphate (3, 2-MeGPP).
The proportion of (-)-2-methylisoborneol (1) decreased from 89 to 17% while that of 2-methylenebornane (4) increased
from 10 to 26%, with the relative yields of the isomeric homo-monoterpenes 2-methyl-2-bornene (5) and 1-methylcamphene
(6) remaining essentially unchanged (o1% each), as determined by chiral GC–MS analysis. The majority of the product mixture
resulting from the MIBS-catalyzed cyclization of 2-MeNPP (10) consisted of the anomalous monocyclic homo-monoterpenes
(
)-2-methylllimonene (15, 39%) and 2-methyl-α-terpineol (13, 10%), as well as the acylic derivatives 2-methylnerol (11, 7%)
and 2-methyllinalool (14, o1%). The steady-state kinetic parameters of the MIBS-catalyzed reaction, determined using
[1-3H]-2-methylneryl diphosphate (2-MeNPP), were kcat 0.0046 0.0003 s À 1, Km 18 6 μM and kcat/Km 2.55 × 102
In comparison, the natural substrate 2-MeGPP had a kcat 0.105 0.007 s À 1, Km 95 49 μM and kcat/Km 1.11 × 103
M
À 1 s À 1
À 1 s À 1
.
M
.
Taken together with earlier X-ray crystallographic studies of MIBS, as well as previous investigations of the mechanistically
related plant monoterpene cyclase, bornyl diphosphate synthase, these results provide important insights into the binding and
cyclization of both native substrates and intermediates and their analogs.
The Journal of Antibiotics advance online publication, 1 March 2017; doi:10.1038/ja.2017.24
INTRODUCTION
(GPP, 2) to form (E)-2-methylgeranyl diphosphate (2-MeGPP, 3).7
Sco7700, 2-MIB synthase (MIBS), catalyzes the Mg2+-dependent
multistep cyclization of the acyclic 2-MeGPP precursor to the
bicyclic alcohol 2-MIB (1) which is accompanied by small
amounts of the bicyclic homo-monoterpenes 2-methylenebornane
(4), 2-methylbornene (5) and 1-methylcamphene (6). The only other
enzyme known to utilize 2-MeGPP as substrate is the closely related
2-methylenebornane synthase (Pfl_1841) of Pseudomonas fluorescens
PfO-1.9 These findings were fully consistent with the results of
independent whole-cell precursor incorporation experiments invol-
ving the feeding of [methyl-13C]methionine and samples of deuterated
mevalonate to the myxobacterium Nannocystis exedens and
MS analysis of the resulting 2-MIB in the head-space volatiles.4
The proposed 2-MIB synthase mechanism is based on close analogy
Since the discovery of 2-methylisoborneol (2-MIB, 1) by Gerber in
1969, there has been considerable interest in the detection, bioreme-
diation and biosynthesis of this volatile, odiferous homo-monoterpene
(Figure 1).1–4 Possessing a musty odor and muddy off-taste, as well as
an extremely low threshold of detection by humans (o10 ng l À 1),
2-MIB is produced by a wide range of Gram-positive actinomycetes,
myxobacteria and cyanobacteria, being second in occurrence only to
the common odiferous sesquiterpene geosmin.5
The elucidation of the biosynthesis of 2-MIB at the molecular
genetic and biochemical level was independently reported in 2008 by
two research groups (Figure 1).6–8 The 2-MIB biosynthetic gene
cluster in Streptomyces coelicolor was shown to harbor a terpene
synthase gene (sco7700) translationally coupled to the gene for an
S-adenosyl-L-methionine-dependent C-methyl transferase, (sco7701), to the well-documented cyclization mechanisms established for a wide
as well as a third gene (sco7699) encoding a protein of still unknown range of monoterpene synthases (Figure 2).10 MIBS initiates the
function, annotated only as a nucleotide-binding protein.7 This electrophilic cyclization by ionizing the Mg2+-complexed substrate
three-gene biosynthetic operon is highly conserved across the genome 2-MeGPP (3) to generate the corresponding allylic cation–pyrophos-
sequences of more than three dozen bacterial species.3,5,6 The phate ion pair which then collapses to the transoid conformer of
S-adenosyl-L-methionine-dependent geranyl diphosphate C-methyl the allylic isomer (3R)-2-methyllinalyl diphosphate (2-MeLPP, 7).
transferase (GPPMT; Sco7701) was shown to catalyze the first Rotation about the C-2,3-bond generates the cisoid conformer of 7,
step in the biosynthetic pathway in S. coelicolor, the electrophilic folded in the anti-endo-boat conformation, which then undergoes
methylation of the olefinic C-2 position of geranyl diphosphate ionization and cyclization to generate the (4R)-2-methyl-α-terpinyl
Department of Chemistry, Box H, Brown University, Providence, RI, USA
Correspondence: Dr DE Cane, Department of Chemistry, Box H, Brown University, Providence, RI 02912-9108, USA.
E-mail: david_cane@brown.edu
This article is dedicated to Professor Satoshi Ōmura, 2015 Nobel Laureate, in Physiology or Medicine, in honor of his profound contributions to basic science and the
improvement of human health as well as his long-standing support of the arts, and in deep appreciation for his continuing support and friendship.
Received 20 October 2016; revised 1 December 2016; accepted 27 January 2017

Incubation of MIBS with the intermediate analog 2-MeNPP
WKW Chou et al
2
Figure 1 Biosynthesis of 2-methylisoborneol (1).
Figure 2 Mechanism of the 2-methylisoborneol synthase (MIBS)-catalyzed
cyclization of 2-methylgeranyl diphosphate (3) to 2-methylisoborneol (1).
A full color version of this figure is available at the The Journal of Antibiotics
journal online.
Figure 3 Mechanism of bornyl diphosphate/α-pinene synthase. A full color
version of this figure is available at the The Journal of Antibiotics journal
online.
cation (8). Electrophilic attack on the cyclohexenyl double bond
of 8 generates the 2-methyl-2-bornyl cation (9), which is then
quenched on the exo face by bound water to generate 2-MIB (1).
Competing deprotonation of intermediate 9 or its proximal rearrange-
ment product readily accounts for formation of the co-products substrate or intermediate analogs.14,15 The 1.80–1.95 Å structures of
4, 5 and 6. The MIBS cyclization mechanism, including the deduced MIBS bound to geranyl-S-thiolodiphosphate and 2-fluorogeranyl
stereochemistry and conformations of the various intermediates, diphosphate, respectively, show each analog coordinated with two
closely resembles that firmly established for (+)-bornyl diphosphate 2 Mg2+ ions and bound in extended conformations in which the
synthase/α-pinene synthase from Salvia officinalis which converts 6,7-double bond of each analog is twisted away from the parallel
GPP (2) to (+)-bornyl diphosphate (75%) as well as a mixture of relationship expected for the postulated anti-endo-boat conformation
the monoterpene hydrocarbons (+)-α-pinene, (+)-camphene, of the actual cyclizing cisoid (3R)-2-MeLPP intermediate (Figure 2).14
(
)-limonene and terpinolene (25% total olefins; Figure 3).11–13 Although this deviation may simply be the result of crystallographic
There are of course two key differences between the reactions trapping of a thermodynamically stable, rather than a kinetically or
catalyzed by MIBS and by (+)-bornyl diphosphate synthase/α-pinene mechanistically relevant conformer, more interestingly it reflects
synthase: (1) MIBS cyclizes 2-MeGPP instead of GPP. Although MIBS the fact that the required 180° rotation of the 2-propenyl substituent
can cyclize GPP to a mixture of monocyclic and bicyclic mono- about the C-2,3 bond of the anti-endo-boat conformer of the
terpenes, the observed kcat is four orders of magnitude lower than that (3R)-2-methyllinalyl diphosphate intermediate is sterically forbidden
for the natural methylated substrate 2-MeGPP.7 (2) The final step by a clash with the 6,7-double bond that is augmented by the presence
in the MIBS-catalyzed cyclization cascade is the quenching of the of the C-2-methyl substituent. It is thus probable that adoption of the
2-methyl-2-bornyl cation exclusively on the exo face by a bound water endo-boat conformation takes place subsequent to conversion of
(Figure 2), while in the (+)-bornyl diphosphate synthase/α-pinene the transoid to the cisoid conformer of 2-MeLPP. Such extended
synthase-catalyzed reaction, the paired pyrophosphate ion is recom- 6,7-double bond conformations of bound substrate analogs have
bined with the bornyl cation exclusively by endo attack (Figure 3).
previously been observed in the structures of a number of canonical
High-resolution crystal structures have recently been determined for monoterpene synthases.16,17 Also thought-provoking were the unex-
MIBS in both an unliganded state and in complex with a number of pected results from co-crystallization of MIBS with racemic
The Journal of Antibiotics

Incubation of MIBS with the intermediate analog 2-MeNPP
WKW Chou et al
3
Figure 4 Synthesis of 2-methylneryl diphosphate (10, 2-MeNPP) and 2-methyl-α-terpineol (13).
2-fluorolinalyl diphosphate (2-FLPP), which had been predicted to
For the planned enzyme incubations, we also needed an authentic
be an unreactive analog of the natural (3R)-2-methyllinalyl reference sample of the homo-monoterpene alcohol ( )-2-methyl-α-
diphosphate intermediate due to the strongly electron-withdrawing terpineol (13),18 which was readily prepared by biomimetically-
2-fluoro substituent. The 2.00 Å structure of the resulting modeled, p-TsOH-catalyzed solvolysis of 2-methylnerol (11) in
complex revealed that the MIBS-bound 2-FLPP had undergone nitromethane (Figure 4).
a reverse allylic rearrangement to generate bound 2-fluoroneryl
diphosphate in complex with 2 Mg2+ ions.15 Also unexpectedly, it In vitro incubation of 2-methylneryl diphosphate with 2-methyliso-
was found that in solution MIBS converted the supposedly unreactive borneol synthase
2-FLPP to camphor, no doubt formed by cyclization of 2-FLPP Before examining the cyclization of 2-MeNPP by methylisoborneol
and spontaneous elimination of hydrogen fluoride from enzymatically synthase, we carried out individual control incubations in the
generated 2-fluoro-2-isoborneol. The observation of enzyme- absence of enzyme with 2-MeGPP and 2-MeNPP and analyzed the
generated 2-fluoroneryl diphosphate trapped within the active site of pentane-soluble extracts by chiral GC–MS in order to identify and
MIBS raises the question whether the cis geometric isomer of 2-FGPP quantify the formation of Mg2+-catalyzed solvolysis products
is a true intermediate of the MIBS-catalyzed ionization and cyclization (Figures 5a and b). The control incubation with 2-MeGPP produced
of 2-FLPP, the nominally unreactive analog of the natural intermediate only racemic ( )-2-methyllinalool (14). The exclusive formation of
2-MeLPP (7), or merely an anomalous shunt metabolite of the this tertiary allylic alcohol contrasts with the well-known Mg2+-cata-
reverse allylic diphosphate rearrangement. We have therefore now lyzed solvolysis of GPP itself, which typically gives a 3:1 mixture of
examined the incubation of MIBS with 2-MeNPP (10), the cis linalool and geraniol.18 The preferred formation of 2-methyllinalool
isomer of the natural substrate, 2-MeGPP (3), and report the (14) by solvolysis of 2-MeGPP presumably reflects the higher free
results below.
energy of 2-methylgeraniol due to the increased steric hindrance about
the tetra-substituted double bond compared to geraniol. In contrast,
the control Mg2+-catalyzed solvolysis of 2-MeNPP gave a mixture of
RESULTS
Preparation of 2-methylneryl diphosphate (10) and ( )-2-methyl- three racemic products, consisting of acyclic ( )-2-methyllinalool
α-terpineol (13) (14) as well as the monocyclic )-2-methyllimonene (15),
(
To prepare 2-MeNPP (11), we used a modification of the previously and ( )-2-methyl-α-terpineol (13; Figure 5b). The Mg2+-catalyzed
reported synthesis of the trans isomer 2-MeGPP (3; Figure 4).7 formation of both ( )-13 and ( )-15 from 2-MeNPP, but not
Wadsworth–Horner–Emmons reaction of 6-methyl-5-hepten-2-one from 2-MeGPP, is consistent with the fact that the (E)-allylic
with triethyl 2-phosphonopropionate followed by LiAlH4 reduction of diphosphate 2-MeGPP (3) is geometrically incapable of direct
the resulting mixture of Z and E conjugated esters gave a 1:1 mixture cyclization. As a positive control, incubation of 2-MeGPP (3) with
of (Z)-2-methylnerol (11) and (E)-2-methylgeraniol (12) which were 2-MIB synthase and chiral GC–MS analysis confirmed the formation
cleanly resolved by flash column chromatography (see Supplementary of ( À )-2-MIB (1) as the major product (89%) accompanied
Information for details). The purified 2-methylnerol (11) was reacted by minor amounts of enantiomerically pure 2-methylenebornane
with triphenylphosphine and CCl4 in an Appel reaction and the (4; 10%), 1-methylcamphene (6; o1%) and 2-methyl-2-bornene
resulting 2-methylneryl chloride was then directly reacted without (5; o1%), as previously observed (Figure 5c and Table 1).6,7
further purification with tris(tetrabutylammonium) hydrogen pyro-
Incubation of 2-MeNPP (10) with MIBS and analysis by chiral
phosphate to give 2-MeNPP (10). The corresponding [1-3H]-2- GC–MS revealed the production of the same group of homochiral
MeNPP ([1-3H]-10) was similarly prepared by oxidation of the bicyclic homo-monoterpenes, ( À )-2-MIB (1, 17%), 2-methylene-
mixture of 2-methylnerol and 2-methylgeraniol to the derived mixture bornane (13, 26%), 1-methylcamphene (6) (o1%) and 2-methyl-2-
of aldehydes, followed by reduction with NaBH3T and chromato- bornene (5) (o1%), but in both substantially different proportions
graphic separation of the resulting mixture of [1-3H]-2-methylnerol and lower overall yield compared to 2-MeGPP (Figures 5d and 6).
and [1-3H]-2-methylgeraniol using AgNO3-impregnated silica gel.
Significantly, four additional, previously unobserved products
The Journal of Antibiotics

Incubation of MIBS with the intermediate analog 2-MeNPP
WKW Chou et al
4
Figure 5 Chiral GC–MS analysis (total ion current, TIC) of incubations with methylisoborneol synthase (MIBS). (a) Control incubation of 2-MeGPP (3) in Mg2
+-containing buffer without MIBS. (b) Control incubation of 2-MeNPP (10) in Mg2+-containing buffer without MIBS. (c) Incubation of MIBS with 2-MeGPP
(3). (d) Incubation of MIBS with 2-MeNPP (10). Previously unobserved product peaks are shown in red. Peaks denoted with *are geraniol internal standard.
A full color version of this figure is available at the The Journal of Antibiotics journal online.
The Journal of Antibiotics

Incubation of MIBS with the intermediate analog 2-MeNPP
WKW Chou et al
5
Table 1 Distribution of products from the incubation of 2-MeGPP (3) Table 2 Steady-state kinetic parameters for total product formation
and 2-MeNPP (10) with 2-MIB synthase
from incubation with methylisoborneol synthase
Product (%)
Substrate
kcat (s À 1
)
Km (μM)
kcat/Km (M À 1 s À 1
)
2-MeGPP (3)
0.105 0.007
95 49
1110
255
Substrate
MIB (1)
4
5
6
13
(
) −15 11
14
2-MeNPP (10)
0.0046 0.0003
18 6
2-MeGPP (3)
89
17
10 o1 o1
26 o1 o1
ND
10a
(10/0)b
ND
39a
(17/22)b
ND
7
ND
o1a
2-MeNPP (10)
DISCUSSION
(0/o1)b
Although (Z)-2-MeNPP (10, 2-MeNPP) shows only a modest fourfold
decrease in the specificity constant kcat/Km compared to the natural
substrate, the trans geometric isomer (E)-2-MeGPP (3), the substantially
altered product distributions resulting from incubation of 2-MeNPP
with 2-MIB synthase establish that 2-MeNPP is a poor surrogate for
2-MeGPP. Thus, while 2-MeNPP could, in principle, be ionized to the
same cisoid allylic cation–pyrophosphate ion pair as the presumptive
intermediate (3R)-2-methyllinalyl diphosphate (7, 2-MeLPP), in
practice incubation of 2-MeNPP with 2-MIB synthase generated a
substantially altered array of bicyclic and monocylic homo-monoterpene
products. Thus, although cyclization of 2-MeNPP generated only a
single enantiomer of each of the four natural bicyclic natural products,
2-MIB (1), 2-methylenebornane (4), 2-methylbornene (5) and
1-methylcamphene (6), the relative proportion of 1 was reduced more
than fivefold from 89% to 17% of the total product mixture, only
partially offset by an increase in the fraction of 2-methylenebornane (4)
which increased from 10 to 26% (Table 1). The majority of the product
mixture resulting from the MIBS-catalyzed cyclization of 2-MeNPP (10)
consisted of the anomalous monocyclic homo-monoterpenes ( )-2-
methyllimonene (15, 39%) and a single enantiomer of 2-methyl-α-
terpineol (13, 10%), as well as the acylic derivatives 2-methylnerol (11,
7%) and a single enantiomer of 2-methyllinalool (14, o1%). Interest-
ingly, while 13, 14 and 15 are not detected among the products of the
in vitro incubation of 2-MIB synthase with 2-MeGPP, all three of these
homo-monoterpenes, of unspecified chiral purity, have been reported as
minor components of the volatile head-space extracts of a small number
of 2-MIB-producing actinomycetes.19,20
Abrreviations: MeGPP, methylgeranyl disphosphate; MeNPP, methylneryl diphosphate;
MIB, methylisoborneol; ND, not detected.
aPercent corrected for background Mg2+-catalyzed solvolysis.
bPercent first eluted peak/percent second eluted peak.
Figure 6 Mechanism of the methylisoborneol synthase (MIBS)-catalyzed
transformation of 2-methylneryl diphosphate (2-MeNPP; 10).
Substantial changes in product distribution have been reported
when the cis isomer neryl diphosphate is incubated with monoterpene
synthases in place of the natural trans allylic substrate, geranyl
diphosphate. For example, in spite of only minor differences in overall
kcat, incubation (+)-BPP/(+)-pinene synthase with NPP instead of
GPP resulted in a decrease in the proportion of the bicyclic
monoterpenes α-pinene and camphene from 79% to 23%, while the
fraction of the monocyclic products limonene and α-terpinolene
constituting 456% of the overall mixture were formed: monocyclic
2-methyllimonene (15, 39%) and 2-methyl-α-terpineol (13, 10%)
along with the acyclic alcohols 2-methylnerol (11, 7%) and 2-methyl-
linalool (14, o1%; Table 1, Figures 5d and 6). Interestingly,
correction for background Mg2+-catalyzed solvolysis of the
2-MeNPP substrate revealed that both 2-methyl-α-terpineol (13)
and 2-methyllinalool (14) were formed as single enantiomers, while
2-methyllimonene (15) was generated as a racemic mixture. The
absolute configurations of the enzymatically generated products 13
and 14 were not determined.
exhibited
a substantial increase from 15 to 71% of total
monoterpenes.21 The enhanced formation of abortive cyclization
products is almost certainly the consequence of aberrant positioning
of 2-MeNPP and NPP in the active sites of the respective homo-
monoterpene or monoterpene synthases, compared to the binding of
the native substrates 2-MeGPP or GPP.
Steady-state kinetics
The fact that cyclization of 2-MeNPP by 2-MIB synthase generates
the naturally occurring enantiomers of 2-MIB (1), 2-methylene-
bornane (4), 2-methylbornene (5) and 1-methylcamphene (6) indi-
cates that at least a portion of this anomalous substrate is bound to
MIBS in a position and conformation that is compatible with,
but certainly not identical to, the precise folding of the natural
substrate 2-MeGPP (3) and the derived intermediate (3R)-2-MeLPP
(7) in the MIBS active site. Although the 2-methylllimonene (15) and
2-methyl-α-terpineol (13) formed from 2-MeNPP are both formally
derivable from a common 2-methyl-α-terpinyl cation, the fact that
15 is generated as a racemic mixture while 13 is produced as
The steady-state kinetic parameters for the MIBS-catalyzed conversion
of [1-3H]-2-MeNPP (10) to total pentane-soluble homo-monoterpene
products were determined and directly compared with those
re-determined for the natural substrate [1-3H]-2-MeGPP (3) under
identical conditions (Table 2). While the turnover number, kcat, for
2-MeNPP (0.0045 s À1) was ~ 20-fold lower than that for 2-MeGPP,
the observed kcat/Km, of 255 M À 1 s À 1 for 2-MeNPP was only 4-fold
lower than that for 2-MeGPP (1105 M À 1 s À 1), reflecting a partially
compensating ~ 5-fold decrease in the observed Km for 2-MeNPP
compared to 2-MeGPP.8
The Journal of Antibiotics

Incubation of MIBS with the intermediate analog 2-MeNPP
WKW Chou et al
6
a single enantiomer indicates clearly that they cannot both be derived 2.38 mmol) was added to the 2-methylneryl chloride in dry CH3CN (10 ml)
under nitrogen and the reaction was stirred at room temperature for 2 days. The
solvent was removed under a gentle stream of N2 to give a viscous, light brown
liquid, followed by the addition of 30 ml of 0.05 M KHCO3 and extraction with
ether (5 × 5 ml) to remove organic impurities. The resulting aqueous phase was
applied to a DEAE-Sephadex column that had been equilibrated with 0.05 M
KHCO3. After loading, the column was washed with 100 ml of 0.05 M KHCO3
and eluted with a linear gradient of 0.05 M KHCO3 (300 ml) to 1.0 M KHCO3
(300 ml) at a flow rate of 2.0 ml min À 1. Fractions (35 ml) were collected and
lyophilized, and those containing 2-MeNPP (10) were dissolved in a minimum
amount of dH2O and applied to a CHP-20P column equilibrated with dH2O.
The column was run with a stepwise gradient of 100 ml of dH2O, 100 ml of 5%
aqueous CH3CN, 100 ml of 10% aqueous CH3CN, 100 ml of 20% aqueous
CH3CN in H2O and 100 ml of 60% aqueous CH3CN in H2O. Fractions were
lyophilized to yield 105 mg of 2-MeNPP (10) as a pale yellow solid (27% yield).
Rf =0.35 (6:3:1 propanol to NH4OH to water). 1H NMR (400 MHz, D2O) δ 5.08
(t, J=4.5 Hz, 1H, H-6), 4.31 (d, J=4.0 Hz, 2H, H-1), 2.05 (m, 2H, H-4), 1.98
(m, 2H, H-5), 1.61 (s, 3H, H-11), 1.58 (s, 3H, H-9), 1.55 (s, 3H, H-10), 1.49
(s, 3H, H-8). 31P {1H} NMR (161 MHz, D2O) δ À 6.6 (d, J=22.5 Hz, Pβ),
À 10.4 (d, J=22.5 Hz, Pα). 13C {1H} NMR (75 MHz, D2O): δ 16.1 (CH3), 17.0
(CH3), 18.2 (CH3), 24.9 (CH3), 26.6 (CH2), 33.3 (CH2), 66.3 (CH2), 124.1 (CH),
133.8 (C), 135.6 (C), 160.3 (C). LC–MS (neg ion ESIMS) m/z 326.99.
from a single-monocyclic carbocation intermediate. The surrogate
2-MeNPP substrate therefore must be bound in at least two distinct
conformations during MIBS-catalyzed cyclization. The fact that only
a single enantiomer of each of the bicyclic, monocyclic and acyclic
alcohols 1, 13 and 14 is produced by incubation of 2-MeNPP with
MIBS also suggests that the same bound water molecule is responsible
for quenching the corresponding carbocation intermediates.
EXPERIMENTAL PROCEDURE
Materials
Reagents and solvents were purchased from Sigma-Aldrich (St Louis, MO,
USA) or Fisher Scientific (Waltham, MA, USA), were of the highest quality
available, and were used without further purification. NaBH3T solution
(6 μmol, 80 Ci mmol À1 specific activity, 500 mCi total in 1 ml of 0.01 N
NaOH) was purchased from American Radiolabeled Chemicals (St Louis, MO,
USA). Isopropylthio-D-galactopyranoside was purchased from Invitrogen
(Waltham, MA, USA). Ni-NTA affinity resin was from Qiagen (Valencia,
CA, USA). Amicon Ultra Centrifugal Filter Units (Amicon Ultra-15 10 000
MWCO) were purchased from Millipore (St Charles, MO, USA). Purified
SCO7700 protein was overexpressed and purified as previously described.7
[1-3H]-2-Methylneryl diphosphate ([1-3H]-10) and [1-3H]-2-
Methods
GC À MS analyses were performed using an Agilent 5977A Series GC/MSD methylgeranyl diphosphate ([1-3H]-3)
A 1.0 ml solution of NaBH3T (6 μmol, 80 Ci mmol À 1, 500 mCi in 1 ml of
0.01 N NaOH) was added to a round bottom flask. The reagent vial was rinsed
with 4 × 1 ml of 0.01 N NaOH which was added to the solution and then placed
under argon, cooled in an ice bath. An ice-cold 1:1 mixture of
2-methylneral to 2-methylgeranial (425 mg, 2.55 mmol), freshly prepared as
previously described,7 in dry EtOH (7.5 ml) was added slowly over 10 min. The
reaction was stirred at 0 °C for 3 h, and the remaining aldehydes were reduced
by the slow addition of unlabeled NaBH4 powder (75 mg, 2.0 mmol). The
resulting solution was warmed to room temperature and stirred overnight.
After a total of 22 h, the reaction was quenched by careful transfer of the
mixture into ice-cold half-saturated aqueous NH4Cl (10 ml). The aqueous layer
was extracted with Et2O (5 × 10 ml) and the combined extracts were dried,
filtered and concentrated to give 310 mg of yellowish liquid (72% crude
chemical yield, 375 mCi, 75% radiochemical yield). The residue was purified
by flash chromatography on 10% (w/w) AgNO3-impregnated silica gel
(EtOAc/hexanes (1:9); 4 × 16-cm column) giving 100 mg of [1-3H]-2-methyl-
nerol ([1-3H]-11, 25% chemical yield, 128 mCi, 26% radiochemical yield) and
210 mg of [1-3H]-2-methylgeraniol ([1-3H]-12, 48% chemical yield, 247 mCi,
49% radiochemical yield). The purified [1-3H]-2-methylnerol ([1-3H]-11) and
[1-3H] 2-methylgeraniol ([1-3H]-12) were individually converted to the
corresponding diphosphate esters using the same method employed for
the synthesis of unlabeled 10 and 3 to yield 98 mg of [1-3H]-2-MeNPP
([1-3H]-10), 37% chemical yield, 6.2 mCi total radioactivity, 2.2% radio-
chemical yield, 28 mCi/mmol specific activity) and 200.1 mg of [1-3H]2-
MeGPP ([1-3H]-3, 36% chemical yield, 105 mCi total radioactivity,
36% radiochemical yield and 232.8 mCi/mmol specific activity). The NMR
spectra of ([1-3H]-10 and of [1-3H]-3 matched those of unlabeled 10 and 3.7
instrument (70 eV, electron impact), a 1-μl injection volume, and a 3 min
solvent delay. Achiral GC À MS conditions used an HP-5 ms capillary
column (0.25 mm ID× 30 m length × 0.25 μm film, Agilent Technologies
(Santa Clara, CA, USA)) and a temperature program with a 2 min hold at
60 °C, a 20 °C min À 1 increment to 280 °C, followed by a 2 min hold at 280 °C.
Chiral GC À MS separations were preformed using a CP-ChiralSil-Dex column
(0.32 mm ID× 25 m length × 0.25 μm film, Agilent) and
a temperature
program with a 1 min hold at 50 °C, a 10 °C min À 1 increment to 200 °C,
followed by a 1 min hold at 200 °C. Compounds detected by GC À MS were
directly compared to their authentic standards using the MassFinder 4.2.1
program (http://www.massfinder.com). Retention indices were measured using
C8–C20 and C10–C40 alkane standards.
LC–MS analyses were performed using a Finnigan LXQ LC–MS instrument
in negative (electrospray ionization ESI) mode, with a Waters Symmetry C18
column (35 μM, 2.1 × 50 mm), a 10 μl injection volume and 1 min signal delay.
The method used was a 1 min isocratic gradient of 95:5 water:acetonitrile, a
gradient to 5:95 water:acetonitrile over 5 min, followed by isocratic elution with
5:95 water:acetonitrile for 9 min. NMR spectra were obtained using a Bruker
1
Avance AV400 NMR spectrometer operating at a 400 MHz H frequency or a
Bruker Avance AV300 NMR spectrometer operating at a 300 MHz 1H
frequency. Liquid scintillation counting was performed using a Beckman-
Coulter LS6500 scintillation counter and Opti-Flour scintillation cocktail from
Perkin-Elmer (Waltham, MA, USA).
All proteins were handled at 4 °C unless otherwise stated. Protein concen-
trations were determined according to the method of Bradford with bovine
serum albumin as the standard.22 Protein purity was estimated using
SDS PAGE gel electrophoresis and visualized using Coomassie Blue stain
according to the method of Laemmli.23
2-Methyl-α-terpineol (13)
2-methylneryl diphosphate (10)
p-TsOH (40 mg, 0.20 mmol) was added to a solution of 2-methylnerol
(200 mg, 1.19 mmol) in 10 ml of MeNO2. The reaction mixture was stirred
at rt for 1 h and monitored by TLC (5:1 hexanes/ethyl acetate). The crude
product was concentrated in vacuo and purified by column chromatography
The cis isomer 2-methylnerol (11) was obtained by preparation of a 1:1 mixture
of 2-methylnerol (11) to 2-methylgeraniol (12) and separation of the isomeric
alcohols using column chromatography as previously described.7 The purified 2-
methylnerol was then converted to 2-MeNPP (10) using the method developed (5:1 hexanes/ethyl acetate) yielding 44 mg of 2-methyl-α-terpineol (13, 22%
by Poulter.24 Triphenylphosphine (1.27 g, 4.75 mmol) was added to a stirring
solution of 2-methylnerol (11; 200 mg, 1.19 mmol) in anhydrous CCl4 (16 ml) alternative method.19 1H NMR (400 MHz, CDCl3) δ 1.98, (m, 2H, H-6), 1.85
and the mixture was refluxed for 24 h. The solvent was evaporated in vacuo and (m, 2H, H-3), 1.72 (m, 1H, H-5), 1.65 (s, 6H, H-10, 11), 1.48 (m, 2H, H-4),
the resulting slurry was triturated with pentane (30 ml) and filtered by suction
filtration. Concentration of the pentane filtrate at room temperature provided
yield). The NMR data matched those previously reported for 13 prepared by an
1.22 (s, 6H, H-8, 9). 13C {1H} NMR (100 MHz, CDCl3) δ 19.4 (C-9, CH3),
19.5 (C-8, CH3), 24.3 (C-3, CH2), 26.3 (C-6, CH2), 27.3 (C-4, CH2),
crude 2-methylneryl chloride, which was used in the subsequent reaction without 32.7 (C-11, CH3), 33.2 (C-10, CH3), 45.8 (C-5, CH), 72.9 (C-7, C),
further purification. Tris(tetrabutylammonium) hydrogen pyrophosphate (1.58 g, 125.0 (C-2, C), 125.8 (C-1, C). GC–MS (EI) m/z 168.1.
The Journal of Antibiotics

Incubation of MIBS with the intermediate analog 2-MeNPP
WKW Chou et al
7
Incubations with 2-methylisoborneol synthase (SCO7700)
Purified 2-MIB synthase (SCO7700; 10 μM) was added to a glass test tube
containing 3.0 ml of assay buffer (50 mM PIPES, 100 mM NaCl, 15 mM MgCl2,
5 mM β-mercaptoethanol and 20% glycerol, pH 6.7) and 60 μM of either
2-MeNPP (10) or 2-MeGPP (3). The enzymatic reaction mixture was overlaid
with 3.0 ml of pentane and incubated at 30 °C for 2 h. The enzymatic products
were extracted with 3 × 3.0 ml of pentane and the organic extracts dried over
Na2SO4, filtered, concentrated in vacuo to 100 μl and analyzed by GC–MS.
Control reactions, measuring the background Mg2+-catalyzed hydrolysis
of each substrate, were conducted similarly as above minus the addition of
SCO7700 to the incubation mixture. An internal geraniol standard (25 pmol)
was added to the organic extracts of the enzymatic and control incubations after
extraction.
3
4
5
6
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& Ikeda, H. Identification and
functional analysis of genes controlling biosynthesis of 2-methylisoborneol. Proc. Natl
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Wang, C. M. & Cane, D. E. Biochemistry and molecular genetics of the biosynthesis of
the earthy odorant methylisoborneol in Streptomyces coelicolor. J. Am. Chem. Soc.
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Steady-state kinetics
Kinetic analyses were performed in 1 ml of assay buffer (50 mM PIPES, 15 mM
MgCl2, 100 mM NaCl, 5 mM β-mercaptoethanol, 20% glycerol and pH 6.7)
with either 2-MeNPP (10, 10–500 μM) and [1-3H]-2-MeNPP ([1-3H]-10,
8.4 Ci/mol) or 2-MeGPP (3, 10–500 mM) and [1-3H]2-MeGPP (([1-3H]-3,
16.5 Ci mol À 1). The reactions were initiated by the addition of 0.20 μM of
SCO7700 protein, overlaid with 1 ml of pentane and incubated at 30 °C for
17 min. The reactions were quenched by the addition of 75 μl of 500 mM EDTA
(pH 8.0) and vortexing for 30 s. The pentane layer was loaded onto a silica
column (2 cm) in a Pasteur pipette and forced through with a stream of
nitrogen into a scintillation vial containing 7 ml of Opti-Fluor. The enzymatic
reaction mixture was extracted 3 more times with 1 ml of ether, and all organic
extracts were passed through the silica column and collected. The combined
extracts were assayed by liquid scintillation counting using a Beckman-Coulter
LS6500 scintillation counter. Kinetic constants were calculated using the
program Kaleidagraph 4.0 and were fit to the Michaelis–Menten equation.
Reported standard deviations in the steady-state kinetic parameters represent
the calculated statistical errors in the nonlinear, least squares regression analysis.
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& Chang, C. C. Biosynthesis of
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CONFLICT OF INTEREST
The authors declare no conflict of interest.
19 Brock, N. L., Ravella, S. R., Schulz, S.
& Dickschat, J. S. A detailed view of
ACKNOWLEDGEMENTS
2-methylisoborneol biosynthesis. Angew. Chem. Int. Ed. Engl. 52, 2100–2104 (2013).
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genome sequenced actinomycetes. Org. Biomol. Chem. 13, 2673–2683 (2015).
This work was supported by NIH grant GM30301 (DEC). We thank Tun-Li
Shen for assistance with MS and Russell Hopson for assistance with NMR
spectroscopy.
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Supplementary Information accompanies the paper on The Journal of Antibiotics website (http://www.nature.com/ja).
The Journal of Antibiotics