A single residue change leads to a hydroxylated product from the class II diterpene cyclization catalyzed by abietadiene synthase

Article abstract of DOI:10.1021/ol3026022

Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotonation to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the class II active site of abietadiene synthase leads to selective production of 8α-hydroxy-CPP instead, demonstrating striking plasticity.

Full text of DOI:10.1021/ol3026022

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A Single Residue Change Leads to a
Hydroxylated Product from the Class II
Diterpene Cyclization Catalyzed by
Abietadiene Synthase
Jared Criswell,† Kevin Potter,† Freya Shephard,‡,§ Michael H. Beale,‡ and
Reuben J. Peters*,†
Department of Biochemistry, Biophysics, & Molecular Biology, Iowa State University,
Ames, Iowa 50011, United States, and Plant Biology and Crop Science Department,
Rothamsted Research, Harpenden, Herts, AL5 2JQ, United Kingdom
rjpeters@iastate.edu
Received September 21, 2012
ABSTRACT
Class II diterpene cyclases catalyze bicyclization of geranylgeranyl diphosphate. While this reaction typically is terminated via methyl deprotona-
tion to yield copalyl diphosphate, in rare cases hydroxylated bicycles are produced instead. Abietadiene synthase is a bifunctional diterpene
cyclase that usually produces a copalyl diphosphate intermediate. Here it is shown that substitution of aspartate for a conserved histidine in the
class II active site of abietadiene synthase leads to selective production of 8r-hydroxy-CPP instead, demonstrating striking plasticity.
The labane-related diterpenoids form a natural products
superfamily, consisting of ∼7000 known compounds, whose
biosynthesis is characterized by (bi)cyclization of (E,E,E)-
geranylgeranyl diphosphate (GGPP; 1), catalyzed via a
protonation-initiated carbocationic cascade reaction
by class II diterpene cyclases.1 The diterpene cyclases
involved in conifer resin acid biosynthesis are bifunctional
enzymes that catalyze such a class II cyclization reaction,
producing CPP (2) from 1 (Scheme 1).2
remains unknown despite previously reported extensive
mutagenesis.4 Given our recent determination of a crystal
structure for the abietadiene synthase from Abies grandis
(AgAS),5 we set out to identify the catalytic base in its class
II active site, resulting in the surprising finding that sub-
stitutions for one potential candidate, histidine 348, led to
mutant AgAS that catalyze class II cyclization reactions
leading to an unusual hydroxylated product.
While the DxDD motif that cooperatively acts as the
catalytic acid is conserved in class II diterpene cyclases, no
conserved residue that might act as the catalytic base has
yet been identified. This may be due to the variation in
the bound substrate configuration implied by the produc-
tion of distinct stereoisomers of CPP by different such
enzymes.1 Accordingly, we were intrigued by the ob-
servation that the class II active site of AgAS contains
Class II diterpene cyclases are characterized by a DxDD
motif, wherein the central aspartic acid residue acts as the
catalytic acid.3 However, the catalytic basefor this reaction
† Iowa State University.
‡ Rothamsted Research.
§ Present address: School of Veterinary Medicine and Science, University
of Nottingham, Sutton Bonington Campus, LE12 5RD, U.K.
(1) Peters, R. J. Nat. Prod. Rep. 2010, 27, 1521.
(2) Keeling, C. I.; Bohlmann, J. Phytochemistry 2006, 67, 2415.
(3) Prisic, S.; Xu, J.; Coates, R. M.; Peters, R. J. ChemBioChem 2007,
8, 869.
(4) Peters, R. J.; Croteau, R. B. Biochemistry 2002, 41, 1836.
(5) Zhou, K.; Gao, Y.; Hoy, J. A.; Mann, F. M.; Honzatko, R. B.;
Peters, R. J. J. Biol. Chem. 2012, 287, 6840.
r
10.1021/ol3026022
XXXX American Chemical Society

Scheme 1. Class II Reactions Mediated by Wild-Type and the
H348D Mutant of AgAS
Figure 1. Class II active site of AgAS (backbone shown in cartoon
format), with the side chains of the aspartates of the DxDD motif
and histidine targeted here shown in stick representation.
metabolic flux to isoprenoids (i.e., 1) in our metabolic
engineering system,14 it was possible to isolate sufficient
amounts of labd-13E-en-8R,15-diol for verification by
NMR spectral analysis (Table S1).
a histidine (at position 348) that is not only appropriately
positioned to act as the baseꢀi.e., across the substrate binding
cavity from the DxDD motif (Figure 1)ꢀbut also further
conserved among the homologous diterpene synthases in-
volved in gymnosperm resin acid biosynthesis, all of which
produce the normal (9S,10S) stereoisomer of CPP (2).
Hypothesizing that this might be the catalytic base, we
substituted alanine for His348 and tested the activity of
the resulting AgAS:H348A mutant in the context of the
D621A substitution that eliminates the subsequently act-
ing class I activity.6 Intriguingly, rather than abolishing
activity, this mutation led to novel products. In particular,
AgAS:H348A/D621A reacted with 1, both in vitro and
in our modular metabolic engineering system (whereby
Escherichia coli is engineered to produce 1 for use by co-
expressed diterpene synthases such as AgAS7), to yield a
hydroxylated product, observed as a dephosphorylated
diol by GC-MS analysis (Figure S1).
Strikingly, a recently reported cis-abienol synthase from
Abies balsamea, homologous to AgAS, similarly proceeds
through 3 as the stable intermediate product of its class II
activity and contains an aspartate in place of the histidine
targeted here, which is otherwise conserved in the known
gymnosperm bifunctional diterpene cyclases.15 Thus, we
constructed the corresponding AgAS:H348D/D621A mu-
tant, which largely produced 3 (Figure 2), rather than the
more evenly distributed mixture of products observed with
AgAS:H348A/D621A. These products included not only 2
and 3 but also a double bond isomer of 2, labda-7Z,13E-
dienyl diphosphate (4), as identified by comparison of the
The mass spectra of this compound resembled that pre-
viously reported for labd-13E-en-8R,15-diol,8 which re-
sults from dephosphorylation of 8R-hydroxy-CPP (3), a
known class II diterpene cyclase product.8ꢀ13 Comparison
with the 3 produced by the relevant enzyme from Nicotiana
glutinosa (NgCLS)9 indicated that AgAS:H348A/D621A
also produces 3 (Figure S1). Moreover, by increasing
(6) Peters, R. J.; Ravn, M. M.; Coates, R. M.; Croteau, R. B. J. Am.
Chem. Soc. 2001, 123, 8974.
(7) Cyr, A.; Wilderman, P. R.; Determan, M.; Peters, R. J. J. Am.
Chem. Soc. 2007, 129, 6684.
(8) Falara,V.;Pichersky, E.;Kanellis,A. K.Plant Physiol. 2010, 154, 301.
(9) Shephard, F. University of Bristol, 2002.
(10) Sallaud, C.; Giacalone, C.; Topfer, R.; Goepfert, S.; Bakaher, N.;
Rosti, S.; Tissier, A. Plant J. 2012, 72, 1–17.
(11) Caniard, A.; Zerbe, P.; Legrand, S.; Cohade, A.; Valot, N.;
Magnard, J. L.; Bohlmann, J.; Legendre, L. BMC Plant Biol. 2012, 12, 119.
(12) Schalk, M.; Pastore, L.; Mirata, M. A.; Khim, S.; Schouwey, M.;
Deguerry, F.; Pineda, V.; Rocci, L.; Daviet, L. J. Am. Chem. Soc.
2012doi: 10.1021/ja307404u.
Figure 2. Effect of H348D mutation on product outcome of the
class II cyclization reaction mediated by AgAS. Chromato-
grams from GC-MS analysis of the dephosphorylated products
(also shown is the structure of 4).
(13) Gunnewich, N.; Higashi, Y.; Feng, X.; Choi, K. B.; Schmidt, J.;
Kutchan, T. M. Phytochemistry 2012doi: 10.1016/j.phytochem.2012.07.019.
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Scheme 2. Roles for Water in Class II Cyclization Reactions
Catalyzed by AgAS or the Mutants Reported Here
Scheme 3. Products of Class I Diterpene Synthases Catalyzed
Reactions with 2 or 3
dephosphorylated alcohol to a previously identified enzy-
matic product (Figure S1).16
It has been indicated that the addition of water in class II
cyclization reactions can only occur when the water mole-
cule is in the optimal position and orientation for addi-
tion to the empty 2p orbital of the relevant carbocation
intermediate, as the water would otherwise serve as a
general base.17 Thus, the ability of AgAS:H348A/D621A
to produce a mixture of compounds resulting from either
direct deprotonation of the labda-13E-en-8-yl carboca-
tion intermediate (5þ) initially formed by bicyclization
(i.e., to yield 2 or 4) or after the addition of water (i.e., to
yield 3) suggests the use of water as a base, at least in this
mutant.
Based on these results, we hypothesize that the histidine
residue found in the wild-type enzyme positions a
water molecule to act as the catalytic base, while
aspartate substitution positions this water for addi-
tion, and the presence of an alanine simply allows
variable positioning. In those cases where water is added,
the resulting oxacarbenium ion must still be deprotonated
to form a stable product, and we further hypothesize that
an additional water molecule in the class II active site may
serve this purpose.
Notably, the observed specific production of the 8R
stereoisomer indicates distal attack of water on the empty
2p orbital of C8 following initial (bi)cyclization (i.e., in5þ).
Such distal addition of water and the observed products
resulting from direct deprotonation (as noted elsewhere
for 2 and 416) are consistent with a concerted cyclization
mechanism (Scheme 2). Although it has been suggested
elsewhere that distal positioning of water for addition is
required for class II cyclization, as a water positioned for
proximal attack might interfere sterically with the preced-
ing cyclization,17 it should be noted that NgCLS has been
suggested to produce a small amount of the 8β-hydroxy
epimer (∼10%),9 hinting at some ability to continue
beyond the initial (presumably concerted) (bi)cyclization,
which would be consistent with the observed production of
rearranged products by class II diterpene cyclases (e.g.,
halimadienyl18 or clerodadienyl19 diphosphates).1
Regardless of the exact mechanism, the ability of
simple substitution of an aspartate for histidine, which
requires only a single nucleotide change, to selectively
change the final product outcome is striking. While the
ability of single residue changes to substantially alter the
product outcome in class I diterpene synthases has been
previously demonstrated,20ꢀ24 the ability of such a change
to alter the chemical composition of the class II cyclization
reaction product was unexpected. Nevertheless, the results
reported here demonstrate intriguing plasticity for at least
the class II diterpene cyclase active sites of the bifunc-
tional diterpene synthases from gymnosperm resin acid
biosynthesis as well. This change further has implications
for the derived diterpenoids (Scheme 3), as the exocyclic-
8(17)-double bond of 2 is involved in the subsequent
cyclization typically catalyzed by the downstream class I
diterpene synthasesꢀe.g., to form pimaradienes (e.g., 6)
and abietanes (e.g., 7). In addition, while class I terpene
synthases also can add hydroxyl groups (e.g., in the for-
mation of725), the presenceofa hydroxyl in3 offersfurther
chemical diversityꢀe.g., the direct production of cis-abienol
(8)15 or a diol such as sclareol (9).11,12
Acknowledgment. This work was supported by a grant
from the NIH (GM076324) to R.J.P., while F.S. was sup-
ported by a BBSRC CASE studentship to M.H.B. and
Quest International. We thank Dr. Jiachen Zi (Iowa State
(18) Nakano, C.; Okamura, T.; Sato, T.; Dairi, T.; Hoshino, T.
Chem. Commun. (Camb.) 2005, 1016.
(19) Hamano, Y.; Kuzuyama, Y.; Itoh, N.; Furihata, K.; Seto, H.;
Dairi, T. J. Biol. Chem. 2002, 277, 37098.
(20) Xu, M.; Wilderman, P. R.; Peters, R. J. Proc. Natl. Acad. Sci.
U.S.A. 2007, 104, 7397.
(21) Wilderman, P. R.; Peters, R. J. J. Am. Chem. Soc. 2007, 129,
15736.
(14) Morrone, D.; Lowry, L.; Determan, M. K.; Hershey, D. M.; Xu,
M.; Peters, R. J. Appl. Microbiol. Biotechnol. 2010, 85, 1893.
(15) Zerbe, P.; Chiang, A.; Yuen, M.; Hamberger, B.; Draper, J. A.;
Britton, R.; Bohlmann, J. J. Biol. Chem. 2012, 287, 12121.
(16) Mafu, S.; Hillwig, M. L.; Peters, R. J. ChemBioChem 2011, 12,
1984.
(22) Morrone, D.; Xu, M.; Fulton, D. B.; Determan, M. K.; Peters,
R. J. J. Am. Chem. Soc. 2008, 130, 5400.
(23) Keeling, C. I.; Weisshaar, S.; Lin, R. P. C.; Bohlmann, J. Proc.
Natl. Acad. Sci. U.S.A. 2008, 105, 1085.
(24) Zhou, K.; Peters, R. J. Chem. Commun. (Camb.) 2011, 47,
4074.
(17) Kolesnikova, M. D.; Obermeyer, A. C.; Wilson, W. K.; Lynch,
D. A.; Xiong, Q.; Matsuda, S. P. Org. Lett. 2007, 9, 2183.
(25) Keeling, C. I.; Madilao, L. L.; Zerbe, P.; Dullat, H. K.; Bohlmann,
J. J. Biol. Chem. 2011, 286, 21145.
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University) for carrying out NMR analysis of labd-13E-
en-8R,15-diol.
Descriptionofexperimental methods. NMR chemical shift
data for labda-13E-en-8R,15-diol. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
Supporting Information Available. Additional figure
illustrating AgAS:H348A/D621A product analysis.
The authors declare no competing financial interest.
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