Dual role for phenylalanine 178 during catalysis by aristolochene synthase

Article abstract of DOI:10.1039/b407932a

A mutant of aristolochene synthase, in which Phe 178 was replaced by Val, produced significant amounts of α- and β-farnesene as well as α and β-selinene and selina-4,11-diene, suggesting that Phe 178 is involved in the stabilisation of transition states preceding germacrene A and following eudesmane cation.

Full text of DOI:10.1039/b407932a

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Dual role for phenylalanine 178 during catalysis by aristolochene
synthase{
Silvia Forcat and Rudolf K. Allemann*
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, UK B15 2TT.
E-mail: r.k.allemann@bham.ac.uk; Fax: 144 121 414 7871; Tel: 44 121 414 4359
Received (in Cambridge, UK) 7th June 2004, Accepted 30th June 2004
First published as an Advance Article on the web 6th August 2004
A mutant of aristolochene synthase, in which Phe 178 was
positive charge on C3 of eudesmane cation. In addition, Phe 178
appeared ideally placed to stabilise carbocations on C2 and C1.
Indeed, stabilisation of the developing positive charge on C1 by Phe
178 in the first step of catalysis had been suggested previously.11
The conversion of FPP to germacrene A proceeds in a stepwise
fashion by way of the intermediate farnesyl cation 5 (Scheme 2).6,8
To address the function of Phe 178 during AS catalysis, we have
produced ASF178Y and ASF178V, in which Phe 178 was replaced
by tyrosine and valine, respectively.
cDNAs for ASF178Y and ASF178V were generated by site
directed mutagenesis from a cDNA of wild type AS which had
been isolated from P. roqueforti. The mutants were expressed to
high levels in BL21(DE3)pLysS cells and purified to apparent
homogeneity.8 The hexane extractable products of the conversion
of FPP by the mutant enzymes were analysed by GC-MS.12 AS
had previously been reported to produce y92% aristolochene (2),
y8% germacrene A (3) and a small amount of valencene (6), which
resulted from deprotonation from C6 rather than C8 in the final
step of the reaction.6 A similar distribution of products was found
for ASF178Y, which converted FPP into y86% 2, y11% 3 and
y3% 6 (Table 1). The steady state kinetic parameters of ASF178Y
were determined by incubation with [1–3H]-FPP and monitoring
the formation of tritiated, hexane extractable products.13 The KM
value of 5.1 mM for ASF178Y was similar to that reported pre-
viously for the wild type enzyme (Table 1). The turnover number
was reduced approximately 30-fold resulting in an overall reduction
of the catalytic efficiency, kcat/KM, of almost two orders of
magnitude. The p-hydroxybenzene ring of tyrosine in ASF178Y
could clearly substitute for the benzene ring of phenylalanine and
direct the substrate along the reaction coordinate to generate close
to wild type products, albeit with reduced speed.
When a valine residue replaced Phe 178, the catalytic efficiency
of the resulting mutant enzyme, ASF178V, was reduced by a
further two orders of magnitude, mostly due to a reduction in the
turnover number (Table 1). The KM of ASF178V was 11.2 mM and
the kcat 2.1 6 1025 s21. Analysis of the hexane extractable products
revealed that the presence of the isopropyl side chain in place of the
aromatic ring of Phe 178 led to the formation of only 10.8% 2
(Table 1). (S)-Germacrene A (3), the enantiomer produced by wild-
type AS, made more than 50% of the total amount of products.
Its stereochemistry was determined through the analysis of the
configuration of the b-elemene produced in the heat-induced Cope
rearrangement of germacrene A by enantioselective gas-chromato-
graphy14 and comparison with (1)-b-elemene, obtained from
the incubation of AS with FPP, as well as a racemic sample of
b-elemene.{
replaced by Val, produced significant amounts of a- and
b-farnesene as well as a and b-selinene and selina-4,11-diene,
suggesting that Phe 178 is involved in the stabilisation of
transition states preceding germacrene
eudesmane cation.
A and following
Aristolochene synthase (AS) from Penicillium roqueforti catalyses
the Mg21-dependent cyclisation of farnesyl pyrophosphate (1,
FPP) to the bicyclic sesquiterpene (1)-aristolochene (2), the parent
hydrocarbon of a large number of fungal toxins including PR-
toxin.1–4 Biochemical labelling and site directed mutagenesis studies
have supported a mechanism in which FPP initially cyclises to
(S)-germacrene A (3),5–7 which undergoes a further cyclisation to
generate the bicyclic eudesmane cation (4) (Scheme 1). Eudesmane
cation is then converted to 2 through a hydride and a methyl shift
followed by deprotonation from C8. The active site of AS provides
the template for the folding of FPP in a conformation favouring the
formation of 2 and at the same time preventing the formation of
alternate reaction products.8,9 AS promotes cyclisation, methyl and
hydride shifts as well as protonation and deprotonation reactions
and prevents premature quenching of the extremely reactive carbo-
cationic intermediates through exclusion of water.
Recent studies have shed some light on the molecular
mechanisms employed by AS to chaperone the reaction inter-
mediates along only one of many possible reaction pathways with
exquisite specificity. Tyr 92 of AS acts as the active site acid
responsible for the protonation of the C6–C7 double bond of
germacrene A that is necessary for the formation of 4 through
electron flow from the C2–C3 double bond.6,9 Trp 334 also
facilitates the energetically demanding generation of 4 through
interactions between the p-system of its indole ring and the
developing positive charge on C3.10
Inspection of the X-ray structure of AS,11 which was obtained in
the absence of a substrate analogue or inhibitor, suggested that the
p-system of Phe 178 might also contribute to the stabilisation of the
The increased formation of germacrene A by ASF178V
suggested that Phe 178 was involved in the stabilisation of a
transition state following this intermediate. The formation of 5.7%
a-selinene (7), 9.1% b-selinene (8) and 2.1% selina-4,11-diene (9)
was observed (Table 1). These bicylic hydrocarbons, which were
identified from their mass spectra and comparison with those of
authentic samples, resulted from eudesmane cation through proton
loss from C2, C4 and C15 (Scheme 2). The formation of significant
amounts of selinenes by ASF178V suggested that Phe 178 was
involved in the stabilisation of the developing positive charge on C2
during AS catalysis. In addition, the bulkiness of Phe 178 might
Scheme 1 Mechanism for the formation of aristolochene from FPP.
{ Electronic supplementary information (ESI) available: total ion
chromatograms from GC-MS analyses of the products of AS,
ASF178Y and ASF178V catalysis; mass spectra of the products generated
by ASF178Y and ASF178V and those of authentic samples. GC-traces and
reaction schemes for the production of b-elemenes; figure indicating the
relative positions of F178, Y92 and W334 within the active site of AS. See
http://www.rsc.org/suppdata/cc/b4/b407932a/
2 0 9 4
C h e m . C o m m u n . , 2 0 0 4 , 2 0 9 4 – 2 0 9 5
T h i s j o u r n a l i s ß T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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Scheme 2 Products formed by ASF178V.
Table 1 Kinetic parameters of and distribution of products formed by AS, ASF178Y and ASF178V
Kinetic parameters
Product distribution
Enzyme
kcat 6 102/s21
KM/mM
kcat/KM/s21 M21
2
3
6
7
8
9
10
11
AS6
ASF178Y
ASF178V
3 ¡ 2
0.1 ¡ 0.049
0.0021 ¡ 0.0004
2.3 ¡ 0.5
5.1 ¡ 1.4
11.2 ¡ 0.18
13 043 ¡ 2 989
196.1 ¡ 110
1.88 ¡ 0.32
91.5
86.4
10.8
7.5
10.7
54.1
v1
2.7
5.2
5.7
9.1
2.1
9.2
2.7
help promote the 1,2-hydride shift. In the absence of the aromatic
group, the stabilisation of the transition state following 4 appeared
to be reduced, eventually leading to aberrant deprotonation and
formation of a- and b-selinene as well as selina-4,11-diene.
Interestingly, ASF178V also produced relatively large amounts
of the two alicyclic sesquiterpenes (E)-b-farnesene (10) and (E,E)-a-
farnesene (11) (Table 1). 10 and 11 formed approximately 9.2% and
2.7% of the total amount of hexane extractable products (Table 1).
These linear products, which were identified from their mass
spectra and through comparison of their mass spectra and reten-
tion times with those of authentic samples, and the significantly
reduced catalytic activity of ASF178V supported an involvement of
Phe 178 in the cyclisation of FPP to germacrene A. However, it
appeared that in addition to stabilising the developing positive
charge on C1 of farnesyl-cation, which might be, at least in part, the
basis of the reduction of the reaction rate of ASF178V, Phe 178
also controlled the barrier height in a step between farnesyl cation
and germacrene A. When an isopropyl-group replaced the
aromatic side chain of Phe 178 the conversion of farnesyl-cation
to germacrene A was slowed and the production of aberrant 10 and
11 through deprotonation from C4 and C15 increased.
The results reported here together with previously published
data6,8 suggest that the cyclisation of FPP to germacrene A might
involve two cationic intermediates. After expulsion of the pyro-
phosphate group of FPP to generate farnesyl-cation (5), cyclisation
leads to germacryl-cation (12), which carries the positive charge
on C11 (Scheme 2). Proton loss from C12 results in the production
of 3.
In summary, Phe 178 appears to fulfil a dual role during AS
catalysis. The efficient conversion of FPP to germacrene A depends
on the interaction of the positive charges on C1 and C11 with the
p-system of Phe 178. In addition, Phe 178 promotes the conversion
of eudesmane cation to aristolochene through stabilisation of the
developing positive charge on C2 and induction of the hydride shift
from C2 to C3 of 4.
to S. F. and grant 6/B17177 (R. K. A.). We thank Wilfried A. Ko¨nig
for a sample of racemic b-elemene from the liverwort Frullania
macrocephalum.
Notes and references
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12 For preparative incubations, enzymes (25 mM) were incubated with FPP
(100 ml, 10 mM) in 500 ml buffer containing 20 mM Tris (pH 7.5), 5 mM
MgCl2, 5 mM 2-mercaptoethanol and 15% glycerol (Buffer A) for 60 h.
The reactions were stopped by the addition of 100 ml of 100 mM EDTA
(pH 7.25), extracted with n-hexane (3 6 3 ml) and vortexed with silica
(1.5 g). The solvent was removed in vacuo and the concentrated samples
were analysed by GC-MS as described8.
13 ASF178Y and ASF178V were assayed in a total volume of 250 ml
containing 2.5 mM enzyme in Buffer A and [1–3H]-FPP (0.8–180 mM).
After termination of the reactions by addition of 200 ml of 100 mM
EDTA (pH 7.25) the samples were extracted three times with n-hexane
and vortexed with 0.5 g of silica. The combined hexane extracts were
analysed for radioactivity as described8.
14 J.-W. de Kraaker, M. C. R. Franssen, A. de Groot, W. A. Ko¨nig and
H. J. Boumeester, Plant Physiol., 1998, 117, 1381–1392.
This work was supported by the BBSRC through a studentship
C h e m . C o m m u n . , 2 0 0 4 , 2 0 9 4 – 2 0 9 5
2 0 9 5