Geosmin biosynthesis. Mechanism of the fragmentation-rearrangement in the conversion of germacradienol to geosmin

Article abstract of DOI:10.1021/ja077792i

Geosmin (1) is responsible for the characteristic odor of moist soil. Incubation of farnesyl diphosphate (2, FPP) with recombinant Streptomyces coelicolor germacradienol/geosmin synthase generated geosmin (1), germacradienol (3), germacrene D (4), octalin 5, and acetone, which was trapped as 2,2-dimethylthiazolidine (9) by reaction with cysteamine. The acetone was shown to be derived by fragmentation of the 2-hydroxypropyl moiety of germacradienol by incubation with [13,13,13-²H₃]FPP (2a), giving rising to the corresponding derivative of d3-acetone, 9a. GC-MS analysis of the labeling pattern of [6-²H]geosmin (1b) resulting from the incubation of germacradienol/geosmin synthase with [2-2H]FPP (2b) revealed that the H-2 proton on FPP undergoes the predicted 1,2-hydride shift to ring B of geosmin. Both sets of experimental results are consistent with a proposed mechanism of geosmin formation involving protonation-cyclization of germacradienol with loss of the 2-hydroxypropyl moiety as acetone by a retro-Prins fragmentation to give octalin intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water. Copyright

Full text of DOI:10.1021/ja077792i

Published on Web 12/21/2007
Geosmin Biosynthesis. Mechanism of the Fragmentation-Rearrangement in
the Conversion of Germacradienol to Geosmin
Jiaoyang Jiang and David E. Cane*
Department of Chemistry, Box H, Brown UniVersity, ProVidence, Rhode Island 02912-9108
Received October 10, 2007; E-mail: david_cane@brown.edu
Scheme 1. Cyclization of Farnesyl Diphosphate to Geosmin
(-)-Geosmin (1) is a degraded sesquiterpene that is responsible
for the characteristic odor of moist soil and is associated with
unpleasant off-flavors in water, wine, and fish.1 Geosmin is pro-
duced by a number of microorganisms, including most Streptomyces
and several species of cyanobacteria, myxobacteria, and fungi.2
A single 726-amino acid protein in Streptomyces coelicolor
A3(2) catalyzes the Mg2+-dependent cyclization of farnesyl di-
phosphate (2, FPP) to a mixture of germacradienol (3), germacrene
D (4), and geosmin (1),3,4 accompanied by small amounts of
octalin 5.5 The closely related 725-amino acid GeoA protein
of S. aVermitilis with 78% identity and 85% similarity to the S.
coelicolor enzyme catalyzes the identical reaction.6 The S. coelicolor
germacradienol/geosmin synthase is a bifunctional enzyme in
which the N-terminal domain of the protein converts FPP (2) to
germacradienol (3) and 4, while the C-terminal domain catalyzes
the transformation of germacradienol (3) to geosmin (1).7 Both the
N-terminal and C-terminal halves have significant sequence similar-
ity to the well-characterized sesquiterpene synthase, pentalenene
synthase.3a,7,8
The mechanism and stereochemistry of the conversion of FPP
to 3 and 4, which is thought to involve the partitioning of a common
germacradienyl cation intermediate 6, has been investigated in detail
(Scheme 1a).3,4,6,7 Formation of germacrene D (4) results from a
1,3-hydride shift of the original H-1si of FPP.3b The alternative
Scheme 2. Cyclization /Fragmentation of Deuterated FPPs to
Geosmin
formation of germacradienol (3), which involves competing loss
of the H-1si proton of FPP (2), can occur by cyclization of 6 to an
enzyme-bound, trans-fused bicyclic intermediate, isolepidozene (7),
a compound that has been isolated from incubation of FPP with
the S233A mutant of S. coelicolor germacradienol/geosmin syn-
thase.7 Isolepidozene (7) would be converted to germacradienol (3)
by proton-initiated ring opening and capture of the resulting
homoallyl cation by water.4,7
By contrast, the mechanistic details of the subsequent conversion
of germacradienol (3) to geosmin (1) are still incomplete. Inde-
pendent incorporation experiments with labeled mevalonates using
Myxococcus xanthus and Stigmatella aurantiaca support the mech-
anism of Scheme 1a in which proton-initiated cyclization of
germacradienol and retro-Prins fragmentation result in formation
of octalin 5 and release of the 2-propanol side chain as acetone
(8).9 Reprotonation of 5 followed by 1,2-hydride shift of the
bridgehead proton into ring B and quenching of the resulting cation
by water will generate geosmin (1).9 This model is supported by
the isolation of octalin 5 as a coproduct of incubations of FPP with
germacradienol/geosmin synthase.5-7 By contrast, an alternative 1,2-
hydride shift of the same bridgehead hydrogen into ring A of
geosmin during biosynthesis in the liverwort Fossombronia pusilla
has also been proposed, on the basis of incorporations of labeled
mevalonate.10 It has been suggested that this mechanism is also
operative in Streptomyces sp. JP95 (Scheme 1b).10 We now report
evidence that conversion of germacradienol (3) to geosmin (1) by
S. coelicolor germacradienol/geosmin synthase results in the release
of the three-carbon side chain as acetone and involves a 1,2-hydride
shift of the bridgehead hydrogen exclusively into ring B of geosmin.
To detect acetone generated in the formation of geosmin, the
product mixture from incubation of FPP with recombinant S.
coelicolor germacradienol/geosmin synthase was reacted with
cysteamine (Scheme 2a).11 GC-MS analysis confirmed the forma-
tion of 2,2-dimethylthiazolidine (9) which displayed a parent peak
at m/z 117 and a prominent [M-CH3]+ at m/z 102. Control
experiments established that neither geosmin nor acetone was
formed when the protein was first inactivated by boiling. To confirm
the origin of the enzymatically generated acetone, [13,13,13-2H3]-
FPP (2a)12 was incubated with germacradienol/geosmin synthase.
The [2H3-Me]-2,2-dimethylthiazolidine (9a) derived from the result-
ing deuterated acetone showed a molecular ion at m/z ) 120
9
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C O M M U N I C A T I O N S
besides the weak molecular ion (m/z ) 182), two other well-defined
fragments at m/z ) 112 and m/z ) 126 correspond to the parent
rings A and B (Figure 2).9.10 Cyclization of [2-2H]FPP (2b) is
predicted to generate [6-2H]geosmin (1b). The observed site of
deuterium labeling in 1b is consistent with the observed shift from
m/z 112 to 113 of the characteristic ring B fragment ion; while the
corresponding ring A-derived fragment ion from 1b, m/z 126, was
devoid of deuterium (Figure 2a). Most importantly, the mass
spectrum of 1b was indistinguishable from that of [6-2H]geosmin
derived from (1R)-[1-2H]FPP, which should differ from 1b only in
the configuration of the C-6 deuterium (Figure 2b).4
The results of conversion of both [13,13,13-2H3]FPP (2a) and
[2-2H]FPP (2b) to geosmins 1 and 1b are fully consistent with the
proposed mechanism of cyclization and fragmentation of germacra-
dienol (3) (Scheme 1a)4,9 while firmly excluding the mechanism
of Scheme 1b10 as well as alternative, mechanistically less likely
proposals.2b The retro-Prins fragmentation that results in the loss
of the germacradienol side chain as acetone has no biochemical
precedent. There is an exceptionally high level of amino acid
sequence conservation (45-78% identity, 57-85% similarity)
among more than a dozen known or presumed microbial geosmin
syntheses.7 The existence of two independent geosmin biosynthetic
pathways, at least among microorganisms, is therefore highly
unlikely.
Figure 1. Mass spectrum of [2H3-Me]-2,2-dimethylthiazolidine (9a).
Acknowledgment. This research was supported by National
Institutes of Health Grant GM30301 to D.E.C.
Supporting Information Available: Experimental methods, incu-
bation conditions, and GC-MS data. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) (a) Gerber, N. N. CRC Crit. ReV. Microbiol. 1979, 7, 191-214. (b) Buttery,
R. G.; Garibaldi, J. A. J. Agric. Food Chem. 1976, 24, 1246-1247.
(2) (a) Gerber, N. N.; Lechevalier, H. A. Appl. Microbiol. 1965, 935-938.
Gerber, N. N. Tetrahedron Lett. 1968, 2971-2974. (b) Pollak, F. C.;
Berger, R. G. Appl. EnViron. Microbiol. 1996, 6, 1295-1299. (c)
Dickschat, J. S.; Wenzel, S. C.; Bode, H. B.; Muller, R.; Schulz, S.
Chembiochem 2004, 5, 778-787. Dickschat, J. S.; Bode, H. B.; Wenzel,
S. C.; Muller, R.; Schulz, S. Chembiochem 2005, 6, 2023-2033. (d)
Scholler, C. E.; Gurtler, H.; Pedersen, R.; Molin, S.; Wilkins, K. J. Agric.
Food Chem. 2002, 50, 2615-2621. (e) La Guerche, S.; Chamont, S.;
Blancard, D.; Dubourdieu, D.; Darriet, P. Antonie Van Leeuwenhoek 2005,
88, 131-139.
Figure 2. Mass spectra of [6-2H]geosmin (1b) derived from (a) [2-2H]-
FPP and (b) (1R)-[1-2H]FPP.
[M+3]+ with fragment ions at m/z ) 102 and 105 resulting from
loss of the CD3- and CH3- groups, respectively (Figure 1). The
presence of the trideuterated 2-hydroxypropyl moiety in the
intermediate [12,12,12-2H3]-germacradienol (3a) was indicated
by a shift of the molecular ion [d3-M]+ from m/z ) 222 to 225
and a corresponding shift in the base peak from m/z ) 59 to 62
[CH3(CD3)CdOH]+, while the [M - acetone]+ fragment at m/z
164 was unchanged. The mass spectrum of the [12,12,12-2H3]-
germacrene D (4a) coproduct also displayed all the predicted
changes. The mass spectra of the derived geosmin (1, m/z 182)
and octalin (5, m/z 164) confirmed the complete absence of
deuterium label in either of these C12 products.
To explore the fate of the H-2 proton of FPP, the requisite [2-2H]-
FPP (2b) (>99 atom % deuterium) was synthesized from trideu-
teroacetic acid by way of [2,2-2H2]trimethylsilylacetic acid using
a modified Peterson olefination procedure that avoids exchange of
the deuterium label.13 GC-MS analysis of the products resulting
from cyclization of [2-2H]FPP (2b) showed the predicted germacra-
dienol-d1 (3b), germacrene D-d1 (4b), octalin-d1 (5b), and geosmin-
d1 (1b) (Scheme 2b). In the mass spectrum of unlabeled geosmin,
(3) (a) Cane, D. E.; Watt, R. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
1547-1551. (b) He, X.; Cane, D. E. J. Am. Chem. Soc. 2004, 126, 2678-
2679.
(4) Jiang, J.; He, X.; Cane, D. E. J. Am. Chem. Soc. 2006, 128, 8128-8129.
(5) Nawrath, T.; Dickschat, J. S.; Mu¨ller, R.; Jiang, J.; Cane, D. E.; Schulz,
S. J. Am. Chem. Soc. 2007, 129, 430-431.
(6) Cane, D. E.; He, X.; Kobayashi, S.; Omura, S.; Ikeda, H. J. Antibiot.
(Tokyo) 2006, 59, 471-479.
(7) Jiang, J.; He, X.; Cane, D. E. Nat. Chem. Biol. 2007, 3, 711-715.
(8) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 1541-1546.
(9) Dickschat, J. S.; Bode, H. B.; Mahmud, T.; Muller, R.; Schulz, S. J. Org.
Chem. 2005, 70, 5174-5182.
(10) Spiteller, D.; Jux, A.; Piel, J.; Boland, W. Phytochemistry 2002, 61, 827-
834. A similar mechanism has also been proposed for the formation of
(+)-dehydrogeosmin in flower heads of the cactus Rebutia marsoneri.
Cf. Feng, Z.; Huber, U.; Boland, W. HelV. Chim. Acta 1993, 76, 2547-
2552.
(11) Shibamoto, T. J. Pharm. Biomed. Anal. 2006, 41, 12-25.
(12) The [13,13,13-2H3]FPP was synthesized by Dr. P. C. Prabhakaran. Cf.
Cane, D. E.; Tandon, M.; Prabhakaran, P. C. J. Am. Chem. Soc. 1993,
115, 8103-8106.
(13) Arigoni, D.; Cane, D. E.; Shim, J. H.; Croteau, R.; Wagschal, K.
Phytochemistry 1993, 32, 623-631.
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