Linalool dehydratase-isomerase, a bifunctional enzyme in the anaerobic degradation of monoterpenes

Article abstract of DOI:10.1074/jbc.M109.084244

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ～1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mM DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg^-1 for the linalool dehydratase and 410 nanokatals mg^-1 for the geraniol isomerase, with apparent K_m values of 750 μM and 500 μM, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).

Full text of DOI:10.1074/jbc.M109.084244

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2010/07/27/M109.084244.DC1.html
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 40, pp. 30436–30442, October 1, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in
□
S
*
the Anaerobic Degradation of Monoterpenes
Received for publication, December 14, 2009, and in revised form, July 13, 2010 Published, JBC Papers in Press, July 27, 2010, DOI 10.1074/jbc.M109.084244
1
Danny Brodkorb, Matthias Gottschall, Robert Marmulla, Frauke L u¨ ddeke, and Jens Harder
From the Department of Microbiology, Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany
Castellaniella (ex Alcaligenes) defragrans strain 65Phen min-
The monoterpenes are divided into acyclic compounds, such
eralizes monoterpenes in the absence of oxygen. Soluble cell as myrcene (7-methyl-3-methylene-1,6-octadiene) and oci-
extracts anaerobically catalyzed the isomerization of geraniol to mene, monocyclic monoterpenes, e.g. limonene and phellan-
linalool and the dehydration of linalool to myrcene. The linalool drene, and bicyclic monoterpenes, e.g. pinene and sabinene.
dehydratase was present in cells grown on monoterpenes, but These unsaturated hydrocarbons are classified as highly volatile
not if grown on acetate. We purified the novel enzyme ϳ1800- organic compounds. Plants as major producers emit more than
fold to complete homogeneity. The native enzyme had a molec- 100 million tons/year to the atmosphere (6) where they are
ular mass of 160 kDa. Denaturing gel electrophoresis revealed photooxidized and contribute to aerosol formation (7, 8). An
one single protein band with a molecular mass of 40 kDa, which example of physiological function is as defense against herbi-
indicated a homotetramer as native conformation. The aerobi- vores: plants often induce the synthesis of monoterpenes as
cally purified enzyme was anaerobically activated in the pres- repellents upon insect damage (9).
ence of 2 mM DTT. The linalool dehydratase catalyzed in vitro
The mineralization of monoterpenes by aerobic microorgan-
two reactions in both directions depending on the thermody- isms has been studied in detail with Pseudomonas species (10,
namic driving forces: a water secession from the tertiary alcohol 11). The aerobic metabolism depends on oxygenases that cata-
linalool to the corresponding acyclic monoterpene myrcene and lyze hydroxylation reactions with molecular oxygen as co-sub-
an isomerization of the primary allylalcohol geraniol in its ste- strate (12). In the absence of oxygen, alternative biochemical
reoisomer linalool. The specific activities (V ) were 140 nano- pathways have been identified for hydrocarbon-mineralizing
max
؊
1
katals mg for the linalool dehydratase and 410 nanokatals bacteria. Alkanes, e.g. n-hexane, and aromatic hydrocarbons
؊
mg for the geraniol isomerase, with apparent K values of 750 with alkyl substituents, e.g. toluene, are anaerobically activated
1
m
␮
M and 500 ␮M, respectively. The corresponding open reading by glycine radical enzymes, and the radical intermediates add to
frame was identified and revealed a precursor protein with a fumarate, yielding methylalkylsuccinate and benzylsuccinate,
signal peptide for a periplasmatic location. The amino acid respectively (13–15). Molybdenum-containing enzymes anaer-
sequence did not affiliate with any described enzymes. We sug- obically hydroxylate ethylbenzene (16) and cholesterol (17).
gest naming the enzyme linalool dehydratase-isomerase accord-
For monoterpenes, no pathway has been elucidated so far.
ing to its bifunctionality and placing it as a member of a new The anaerobic mineralization of monoterpenes to carbon diox-
protein family within the hydrolyases (EC 4.2.1.X).
ide is frequently present in denitrifying bacteria (18). Cultiva-
tion approaches established the enrichment of monoterpene-
mineralizing microorganisms (19) and the isolation of strains of
Monoterpenes constitute a large and extremely diverse Alcaligenes defragrans (20) and Thauera terpenica (21). A.
group of natural compounds within the isoprenoids (1–3). defragrans was recently placed in the newly defined genus Cas-
They are synthesized from two five-carbon units of isopentenyl tellaniella, as C. defragrans (22). Initial studies on potential
pyrophosphate, a derivative of isoprene. This central interme- metabolites of the degradation pathway identified isoterpi-
diate is formed in two alternative pathways. In the mevalonate- nolene as metabolite that was apparently not further metabo-
dependent route, isopentenyl pyrophosphate is synthesized lized (23) and geranic acid as ionic intermediate present in
from acetyl-CoA via mevalonic acid. It represents an important nitrate-respiring cells that were grown on acyclic or cyclic
cellular metabolic pathway in all higher eukaryotes, Archaea monoterpenes, e.g. myrcene or limonene (24).
and many Bacteria. (E)-4-Hydroxy-3-methyl-but-2-enyl pyro-
A simple pathway hypothesis is a hydration of myrcene, lead-
phosphate is the central precursor in the nonmevalonate path- ing to geraniol and further to geranic acid (Fig. 1). We initiated
way utilized by algae, the plastids of higher plants and some biotransformation studies with soluble extracts of C. defra-
Bacteria (4, 5).
grans. In this article we report on the detection of novel enzyme
activities and the isolation and characterization of an anaerobic
linalool dehydratase-isomerase, a bifunctional enzyme that cat-
alyzes the reversible dehydration and isomerization of linalool
*
This work was supported by the Max Planck Society.
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S3.
□S
(
3,7-dimethyl-1,6-octadien-3-ol) (Fig. 1).
The nucleotide sequence(s) reported in this paper has been submitted to the
TM
GenBank /EBI Data Bank with accession number(s) FR669447.
1
To whom correspondence should be addressed: Dept. of Microbiology, Max EXPERIMENTAL PROCEDURES
Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen,
Reagents—R-Limonene (95%), myrcene (90%), linalool (99%),
Germany. Tel.: 494212028750; Fax: 494212028580; E-mail: jharder@
mpi-bremen.de.
and geraniol (98%) were purchased from Sigma-Aldrich. All
3
0436 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010

Linalool Dehydratase-Isomerase in Monoterpene Degradation
were determined in a one-phase sys-
tem with 10% (v/v) DMSO. The
reaction was started by adding
monoterpene (0.1–10 mM) that was
dissolved in anoxic 80 mM Tris-HCl
buffer, pH 9.0, with 10% DMSO. In a
third assay system, a pure myrcene
phase (1/2 (v/v) myrcene/Tris-HCl
FIGURE1. Proposed anaerobictransformation ofmyrcene in C. defragrans. A, linalooldehydratase-isomer-
ase; B, geraniol dehydrogenase; C, geranial dehydrogenase.
buffer) was applied for measuring
linalool and subsequently geraniol
other chemicals used were of the highest available purity and formation. The tubes were immediately transferred into a 35 °C
were purchased from Aldrich, Boehringer, Fluka (Neu-Ulm, shaking incubator. For kinetic analyses and the myrcene turn-
Germany), Merck, Sigma, and Bio-Rad Laboratories. Gases over, aqueous samples were taken at different time points and
(
CO grade 4.8, N grade 5.0, and O grade 2.0) were supplied by directly injected into the GC. In the two-phase system, 1 ␮l of
2
2
2
Air Liquide (D u¨ sseldorf, Germany). Chromatography media the organic HMN carrier phase was injected to determine the
and instruments were from GE Healthcare.
substrate and product concentration.
Cell Growth and Preparation of Soluble Extracts—C. defra-
To estimate the effect of temperature on linalool dehydratase
grans strain 65Phen was maintained as described (20). For bio- activity, the two-phase assay was performed at temperatures
mass production, the strain was cultivated on 30 mM limonene between 4 °C and 45 °C. The pH optimum was tested by varying
and 100 mM nitrate (24). A 1-liter preculture was inoculated in the buffer systems with pH values near the specific pK values at
a
a 10-liter vessel of carbonate-buffered mineral salt medium at 35 °C. The two-phase assay was also used to determine the
pH 7.0. Filter-sterilized limonene and vitamins (25) were added influence of different effectors on enzyme activities.
after cooling, and the culture was incubated for 6–7 days with a
The concentrations of the monoterpenes were analyzed by GC
Ϫ1
CO /N (10/90 (v/v)) gas stream of 24 ml h at 28 °C. The (Auto System XL; PerkinElmer Life Sciences) equipped with an
2
2
stirrer frequency was initially 150 rpm and was increased dur- Optimaꢀ-5(0.25-␮mfilmthickness, 50m ϫ0.32-mminnerdiam-
ing exponential growth phase of C. defragrans up to 250 rpm to eter; Macherey-Nagel, D u¨ ren, Germany) column and flame ioni-
ensure optimal substrate availability.
zation detector. The following temperature program was applied:
Cell harvest began after the addition of reducing agents, 50 ␮M injection port temperature, 250 °C; column start temperature,
Ϫ1
Fe(II)Cl and 2 mM DTT. Cells in the late exponential growth 85 °C for 1 min, increasing to 120 °C at a rate of 5 °C min , 120 °C
2
Ϫ1
phase (A Ϸ 3) were transferred by gas pressure to centrifuge for 0.1 min, increasing to 290 °C at a rate of 45 °C min , 290 °C for
600
tubesandthencollectedbycentrifugationfor15minat9000ϫgat 1 min; detection temperature, 350 °C. The split ratio was set to
4
°C. For the preparation of the soluble proteins, 40 g of wet or 1:25.
frozen cells were suspended in 60 ml of 25 mM sodium phosphate
Purification of Linalool Dehydratase—The purification was
¨
buffer, pH 8.0, containing 2 mM DTT and disintegrated in two performed with an Akta system (GE Healthcare). All purifica-
passages through a French pressure cell press (Amincon, Roches- tion procedures were carried out at 4 °C with filtered (0.2 ␮m)
ter, NY) at 10.3 MPa. The soluble fraction was obtained by ultra- and degassed buffers. 100 ml of soluble extract obtained from
centrifugation for 90 min at 150,000 ϫ g at 4 °C to remove cell cells grown on limonene was applied to a Source 30Q column
debris, unbroken cells, and membrane proteins.
(5 ϫ 30 cm) equilibrated in 50 mM sodium phosphate, pH 8.0
Assays for Geraniol Isomerization and Linalool Dehydration— (AIE-Q1). The enzyme eluted at 200 mM NaCl in the aforemen-
Salt or urea containing linalool dehydratase fractions were dia- tioned buffer during a stepwise gradient performed with 3.5 ml
Ϫ1
lyzed three times against a 1000-fold volume of 80 mM Tris-HCl min . Fractions containing linalool dehydratase were pooled,
buffer, pH 9.0, for 20 min at 4 °C and under magnetic stirring. and saturated ammonium sulfate solution was added to a final
Purified and dialyzed linalool dehydratase fractions were stored concentration of 15% (v/v). The protein solution was applied to
under an anoxic gas phase at 4 °C.
a Butyl-Sepharose FF column (4.7 ml) preequilibrated with 15%
Geraniol isomerization and linalool dehydration were as- (v/v) saturated ammonium sulfate in 80 mM Tris-HCl, pH 8.0.
sayed routinely in a two-phase system. Vials (17 ϫ 38 mm; After a first elution with 80 mM Tris-HCl, pH 8.0, the target
Zinsser Analytic, Frankfurt, Germany) were prewarmed at enzyme was eluted with 6 M urea. The urea fraction, typically 20
3
5 °C. Anoxic protein solution was transferred into the vials, ml, was mixed with saturated ammonium sulfate solution to a
and DTT was added to 2 mM. The tests were sealed with a butyl final concentration of 40% (v/v). After centrifugation for 10 min
septum, and the headspace was flushed with CO /N (10/90 at 20,000 ϫ g and 20 °C, the supernatant was withdrawn, and
2
2
(
v/v)). The reaction was started by adding a distinct linalool or the pellet was solved in 2 ml of 100 mM Tris-HCl, pH 8.0. The
TM
geraniol concentration to investigate the reaction to myrcene. concentrated solution was passed through a Superdex
1
0–100 mM organic substrate was dissolved in 2,2,4,4,6,8,8- 200-pg column (120 ml) equilibrated with 10 mM Tris-HCl, pH
2
heptamethylnonane (HMN). The organic phase was added in a 9.0. The active fractions from the gel filtration were applied to a
1
:1 ratio to the aqueous protein solution. Kinetic parameters second anion exchange chromatography with a different col-
umn material (ResourceQ, 1 ml) that was preequilibrated with
2
10 mM Tris-HCl, pH 7.0. The enzyme eluted at 120 mM NaCl in
The abbreviations used are: HMN, 2,2,4,4,6,8,8-heptamethylnonane; DMSO,
Ϫ1
dimethyl sulfoxide.
a step gradient performed with 2 ml min (AIE-Q2).
OCTOBER 1, 2010•VOLUME 285•NUMBER 40
JOURNAL OF BIOLOGICAL CHEMISTRY 30437

Linalool Dehydratase-Isomerase in Monoterpene Degradation
Determination of Relative Molecular Mass—The apparent
relative molecular mass of the native enzyme was determined
TM
by gel filtration on a Superdex 200-pg column (120 ml) in 80
mM Tris-HCl buffer, pH 9.0, The standard proteins were: cata-
lase (232 kDa), aldolase (158 kDa), bovine serum albumin (67
kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa).
The molecular mass of the monomeric enzyme was determined
using a 12% SDS-polyacrylamide gel stained with Coomassie Blue
R-250 (26). The protein ladder (Fermentas, St. Leon-Rot, Ger-
many) covered a range molecular masses from 10 to 170 kDa.
UV-Visible Spectroscopy—Standards and purified linalool
dehydratase fractions were dissolved in 80 mM Tris-HCl, pH
Ϫ1
9
.0, in the range between 10 and 100 ␮g ml . UV absorption
spectra were obtained by using a DU 600 UV-visible spectro-
photometer (Beckman Coulter, Krefeld, Germany).
Protein Determination—The protein content was measured
by Coomassie Blue R-250 protein assay (27) and by using bovine
serum albumin as the standard.
N-terminal Amino Acid Sequence Analysis—Purified linalool
dehydratase was separated by 12% SDS-PAGE and electro-
blotted on a PVDF membrane (Sequi-Blot; Bio-Rad Laborato-
ries) according to the method of Towbin et al. (28). The mem-
brane was washed for 1 min with distilled water and stained with
Coomassie Blue R-250 (0.025% (v/v) in 40% (v/v) methanol) for
FIGURE 2. SDS-PAGE of the linalool dehydratase-isomerase after purifi-
cation. The sizes of marker proteins are indicated in kDa. Gels with 7.5%
acrylamide revealed the absence of smaller proteins (data not shown).
30 s before destaining with a water/methanol/acetic acid mixture
(50/45/5, v/v/v). The PVDF membrane was washed with distilled
water and dried for 6 h. The protein was excised from the mem- alool dehydratase activities. In contrast, the enzyme activity
brane, and Edman degradation of the N-terminal amino acid res- was not detected in cells grown on acetate. Addition of limo-
idues was performed by Toplab GmbH (Martinsried, Germany). nene (10 mM) to the culture growing on acetate resulted in
The gene and the protein sequence were deposited at GenBank induction of the enzyme activity after 10 h (data not shown).
under accession no. FR669447.
Purification of Linalool Dehydratase from C. defragrans
Overexpression in Escherichia coli—Standard molecular biol- Strain 65Phen—The purification of the linalool dehydratase ini-
ogy methods were applied. In short, the ldi gene was amplified tially yielded a preparation with several proteins (data not shown).
with the primers ldi_NdeI_fw (TGCGACATATGATGCGGT- The purification procedure was significantly improved by includ-
TCACATTG) and ldi_BglII_rw (CGCGAGATCTTTATTTC- ing a Butyl-Sepharose column. The protein eluted from this col-
CCTGCGA) from genomic C. defragrans DNA and ligated into umn with 6 M urea. In a five-step protocol, the enzyme activity was
pCR4-TOPO (Invitrogen). The NdeI-BglII-flanked gene was purified to a single protein band (Fig. 2). The linalool dehydratase
transferred into pET-42a(ϩ) (Novagen, Merck KGaA), and the protein yield was 0.02% of the initial protein, accompanied by a
TM
gene was expressed in E. coli BL21 Star (DE3) (Invitrogen). The 1846-fold increase in the specific activity (Table 1). Gel filtration
TM
construct correctness was confirmed by sequencing. Cultures chromatography on a Superdex 200-pg column gave a single
were induced with isopropyl 1-thio-␤-D-galactopyranoside. Solu- peak of active protein after a retention volume of 62 ml. Based on a
ble extracts were assayed in the anaerobic two-phase system.
calibration with standard proteins, linalool dehydratase exhibited
a native molecular mass of 160 kDa. SDS-PAGE of the purified
enzyme showed a single band with a molecular mass of 40 kDa
RESULTS
Soluble extracts of C. defragrans catalyzed the transforma- (Fig. 2). These observations suggested that the native form of lin-
ϩ
tion of geraniol in two directions (Fig. 1). A NAD -reducing alool dehydratase from C. defragrans 65Phen is likely a homotet-
3
activity showed the presence of a geraniol dehydrogenase. In ramer (␣ ). UV-visible absorbance spectra revealed the absence of
4
the absence of an electron acceptor, the dialyzed soluble extract chromophors between 300 and 850 nm, suggesting that there was
initially formed linalool, and then, after a certain linalool con- no prosthetic group present.
centration was reached, myrcene appeared. Both compounds,
Catalytic Properties of Purified Linalool Dehydratase—The
linalool and myrcene, were detected and identified by GC and purified protein catalyzed the dehydration of linalool to myr-
GC-MS (data not shown). In separate experiments, the dialyzed cene in the absence of molecular oxygen but required 2 mM
soluble extract transformed linalool to myrcene.
DTT. Geraniol was isomerized initially to linalool, and subse-
Biomass yields in a pH-controlled fermenter were lower on quently myrcene appeared (Fig. 3A). Both activities occurred
myrcene than on limonene. Hence, we grew C. defragrans on concurrently in all purification steps; e.g. Fig. 4 shows the final
limonene. The crude extracts showed comparable specific lin- anion exchange purification. The purification of the geraniol
isomerase activity was 1740-fold similar to the 1846-fold puri-
3
J. Harder, unpublished results.
fication of the linalool dehydratase activity.
3
0438 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010

Linalool Dehydratase-Isomerase in Monoterpene Degradation
TABLE 1
Purification of linalool dehydratase from C. defragrans 65Phen
Purification step
Protein
Activity
Specific activity
Relative specific activity
Protein yield
mg
nanokatals
nanokatals/mg
%
Soluble extract
2490.3
401.6
33.3
15.3
6.9
12.9
25.5
19.2
33.9
19.3
5.3
0.005
0.063
0.575
2.213
2.783
9.228
1
13
100
16.13
1.34
0.61
0.28
0.02
Anion exchange (AIE-Q1)
Hydrophobic interaction
Ammonium sulfate precipitation
Size exclusion
115
443
557
1846
Anion exchange (AIE-Q2)
0.6
carrier nor in a DMSO-containing
aqueous system. Myrcene was the only
product detected in these experiments.
We tested other acyclic monoter-
penes as substrate for the enzyme.
Neither the monoterpenes ␣- and
␤
-ocimene nor the monoterpenoids
citronellol and nerol were trans-
formed. A 3-methylene group is
absent in the ocimenes that have a
3-methyl-1,3-diene structure. Of the
cis-3-methyl-2-en-1-ol motif present
in geraniol, citronellol lacks the dou-
ble bond at the C2-carbon atom, and
nerol is the trans-isomer to geraniol.
This suggests ahighly specificbinding
FIGURE 3. Time course of monoterpene transformation by the purified linalool dehydratase-isomerase
inatwo-phase-systemwithHMNasorganiccarrierphase(A)andinthepresenceofamyrcenephase(B). site for the substrates.
ꢁ
, myrcene; f, linalool; Œ, geraniol.
Effects of Various Compounds—The
purified linalool dehydratase required
only DTT as a reducing agent and an
The enzyme activity measurements were performed in a two- oxygen free microenvironment (Ͻ1% (v/v)) for the dehydration of
phase system with HMN as organic phase. Like other monoterpe- linalool. The activity was not detectable in the presence of 1 mM
nes, myrcene is 100-fold less soluble in water than monoterpe- Ti(III)citrate. Other inhibitors were molecular oxygen (Table 2)
noids, e.g. geraniol or linalool: myrcene has a solubility of 43 ␮M and high salt concentrations. NaCl, KCl, or MgCl at a concentra-
2
and a octanol/water partition coefficient of logP ϭ 4.5 (29). The tion of 220 mM inhibited the enzyme activity completely. The met-
organic phase served also as reservoir for the monoterpenoids. al-chelating agent EDTA (5 mM) did not affect the enzyme activity,
This dilution influences the actual concentrations of geraniol and suggesting that either the protein does not require metal ions for
linaloolinaqueoussolution. Inequilibriumwiththeorganicphase, activity or the chelating molecule was not able to remove the metal
calculation revealed micromolar concentrations for geraniol and ions under the assay conditions. Potassium nitrite or nitrate (20
linalool in the aqueous phase. Observed rates under these condi- mM) did not influence the enzyme activity. Coenzyme A was inef-
Ϫ1
tions were low, 14.5 picokatals mg for linalool dehydratase and fective as a cofactor. However, phosphate as buffer or pyridoxal
Ϫ1
8
.8 picokatals mg for geraniol isomerase.
phosphate as well as S-adenosylmethionine modulated the
The enzyme activities were not inhibited by 10% (v/v) DMSO. enzyme activity. The enzyme is inhibited by urea: 20% activity
Thus, we performed the kinetic characterization in a single-phase remained at 3 M urea, and no activity was detected in 6 M urea.
system with 10% (v/v) DMSO in water. The linalool dehydratase
Optimal pH and Thermophilicity—The linalool dehydratase
and the geraniol isomerase activities exhibited typical Michaelis- activity had an optimal temperature at 35 °C. The enzyme activity
Ϫ1
Menten kinetics with V values of 140 and 410 nanokatals mg
had a pH maximum at low alkaline conditions (supplemen-
max
protein, respectively (Fig. 5). The K values for linalool and gera- tal Fig. S1), but there was a sharp decrease in linalool dehydratase
m
niol were 750 ␮M and 500 ␮M, respectively.
activity beyond pH 9.0. The optimal pH was 9.0 with Tris-HCl
Quantification of the reverse reactions, the hydration of myr- buffer. The temperature dependence of the reaction showed a lin-
cene to linalool and the isomerization of linalool to geraniol, were ear Arrhenius plot in the range from 22 °C to 35 °C (supple-
attempted with a myrcene-saturated aqueous phase that was mental Fig. S2), with an activation energy of E ϭ 68.6 kJ/mol.
A
maintained by a pure myrcene phase. The formation of linalool
Identification of the Open Reading Frame—The N-terminal
proceeded initially with a maximum specific activity of 133 protein sequence was determined, and the corresponding open
Ϫ1
picokatals mg (Fig. 3B). After an accumulation of 0.2 mM linal- reading frame was found within a fosmid sequence obtained
4
ool, geraniol was formed at a similar rate. This experiment from C. defragrans 65Phen. The gene coded for a preprotein
revealedthereversibilityoftheenzymeactivity. Insystemswithout
a pure myrcene phase, we never detected the formation of geraniol
4
from linalool, neither in the two-phase system with an organic
A. W u៮ lfing, F. Germer, A. Meyerdierks, and J. Harder, unpublished results.
JOURNAL OF BIOLOGICAL CHEMISTRY 30439
OCTOBER 1, 2010•VOLUME 285•NUMBER 40

Linalool Dehydratase-Isomerase in Monoterpene Degradation
with 397 amino acids, including an N-terminal signal peptide eukaryotic ascomycota Aspergillus oryzae RIB40 with an E
sequence (MRFTLKTTAIVSAAALLAGFGPPPRAA) for trans- value of 2E-08. The biotransformation potential of Aspergillus
port into the periplasmatic space (supplemental Fig. S3). The ala- species on myrcene has been elucidated previously (33),
nine pair residues represent the cleavage motif. The experi- although linalool was not detected as a transformation product.
mentally determined N terminus of the purified protein TblastP identified a hypothetical protein from another eukary-
started with the second alanine of the predicted cleavage otic ascomycota, Nectria hematococca mpVI, as closest related
motif (AELPPGRLATTE). Analyses with SignalP 3.0 (30) protein, with an E value of 2E-12.
based on studies of signal-sequence cleavage sites (31) sug-
A ClustalW alignment of the linalool dehydratase-isomerase
gested a Sec-dependent membrane translocation mecha- showed no relevant scores with characterized alkene hydrata-
nism for the preprotein into the periplasmatic space. Thus, ses, namely a ␥-carotene 1,2-hydratase (CruF) from Deinococ-
the purified protein represents a mature protein. According cus radiodurans R1 (34), a hydroxyneurosporene synthase from
to in silico mass calculation the precursor exhibits a molec- Rhodospirillum rubrum ATCC 11170, and a ␥-carotene 1,2-
ular mass of 43 kDa.
hydroxylase from Synechococcus sp. PCC 7002. This was a fur-
Comparisons of the protein and of the gene sequences with ther indication of the novel character of this enzyme and its
nucleotide, microbial genome and environmental met- catalytic activity.
agenomic datasets did not reveal significant relationships to
Expression of Linalool Dehydratase-Isomerase in E. coli—
known proteins and genes. TblastN (32) identified the closest The identified open reading frame was used to construct the
relative as a hypothetical partial mRNA protein from the expression vector pET-42a(ϩ)-LDI. Isopropyl 1-thio-␤-D-ga-
lactopyranoside-induced 3-ml cultures showed a linalool dehy-
dratase activity of 380 nanokatals and a geraniol isomerase
activity of 310 nanokatals in a 6-h assay. Control cultures with
the vector lacking the ldi gene had no enzyme activity. Sol-
uble extracts of the induced cells had a specific linalool dehy-
Ϫ1
dratase activity of 435 picokatals mg protein and a gera-
Ϫ1
niol isomerase activity of 116 picokatals mg protein in the
two-phase assay.
DISCUSSION
Myrcene is an acyclic C -hydrocarbon and represents a
1
0
large fraction (74%) of monoterpenes extracted from the essen-
tial oils of the hop plant Humulus lupulus (35). The transfor-
mation of this unsaturated hydrocarbon at the enzymatic level
FIGURE 4. Enzyme purification by anion exchange chromatography (AIE-
Q2). Linalool dehydratase and geraniol isomerase activities eluted together
at 120 mM sodium chloride. The most active fraction contained a single pro- has never investigated under anaerobic conditions. Here, we
tein on SDS-PAGE (Fig. 2).
describe a new initial reaction in the
anaerobic degradation of hydrocar-
bons: the hydration of myrcene.
First, a water molecule is added to
the methylene double bond. Mech-
anistically, it may be equivalent to a
chemical water addition catalyzed
by acids, leading to linalool, a terti-
ary allylalcohol (3,7-dimethyl-1,6-
octadien-3-ol). A subsequent isom-
erization yielded the primary
allylalcohol
geraniol
(3,7-di-
methylocta-2,6-dien-1-ol). These
two reactions are catalyzed by a
single bifunctional enzyme, the
linalool dehydratase-isomerase.
The thermodynamic equilibrium
favors the formation of myrcene
from geraniol. Linalool is thermo-
dynamically more stable than gera-
niol: according to experimental
observations in a two-phase system
with Thauera linaloolentis (36), lin-
Ϫ1
alool is 5.9-kJ mol
more stable
FIGURE 5. A and B, Michaelis-Menten plots of linalool dehydratase activity (A) and geraniol isomerase activity
B). C and D, Hanes plots revealing the Km and Vmax values. pkat, picokatals.
(
than geraniol. Our observations con-
3
0440 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 40•OCTOBER 1, 2010

Linalool Dehydratase-Isomerase in Monoterpene Degradation
TABLE 2
Effectors on enzyme activity
involves a cytosolic preprotein which, typical for these translo-
cated proteins, has a short signal sequence with a two alanine
The complete assay contained 150 ␮l of 100 mM monoterpene dissolved in HMN
motif at the end, representing the cleavage site (31). The pre-
protein is transported across the cytoplasmatic membrane in
an unfolded state. During this process, the signal peptide is
cleaved. A periplasmatic location for a hydrocarbon activation
enzyme was already detected in the denitrifying Azoarcus strain
EbN1 (38). A periplasmatic dehydrogenase oxidizes ethylben-
zene to (S)-1-phenylethanol. Future experiments with protein
labeling or specific antibodies may describe the translocation in
more detail.
and 150 ␮l of protein solution (0.5 mg/ml) including 2 mM DTT.
Assay
Linalool dehydratase Geraniol isomerase
%
%
Complete assay
100
10
0
100
Ϫ2 mM DTT
10
ϩ1 mM Ti(III)citrate
0
ϩ0.1% (v/v) O
100
110
90
5
100
110
90
2
2
ϩ0.5% (v/v) O
ϩ1% (v/v) O
2
ϩ20% (v/v) O
5
2
ϩ99.9% (v/v) O
0
0
2
ϩ1 mM pyridoxal phosphate
65
20
100
200
In summary, this work depicts the enzyme for the initial
metabolism of myrcene, an acyclical monoterpene, under
anaerobic conditions. It is a novel type of dehydratase-isomer-
ϩ40 mM S-adenosylmethionine
firmed this equilibrium with the enzyme from C. defragrans: the ase that acts on myrcene, linalool, and geraniol. We recom-
linalool dehydratase-isomerase catalyzes the formation of linalool mend the disposition of a new protein family with the EC num-
from geraniol and subsequently of myrcene from linalool. In this ber 4.2.1.X.
thermodynamically favorable direction, the formation of myrcene
fromgeraniolmaybeseenasdetoxificationprocessforthemonot- Acknowledgment—We thank Hannah Marchant for improvement of
erpene alcohol. The monoterpene alcohols have a higher cell tox- the language.
icity than the monoterpenes.
The observation of the reverse reactions, myrcene to linalool
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