Plant carotene cis-trans isomerase CRTISO: A new member of the FAD red-dependent flavoproteins catalyzing non-redox reactions

Article abstract of DOI:10.1074/jbc.M110.208017

The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-α/ β-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates.Thereaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene β-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction.

Full text of DOI:10.1074/jbc.M110.208017

Enzymology:
Plant Carotene Cis-Trans Isomerase
CRTISO: A NEW MEMBER OF THE
FADRED-DEPENDENT
FLAVOPROTEINS CATALYZING
NON-REDOX REACTIONS
Qiuju Yu, Sandro Ghisla, Joseph Hirschberg,
Varda Mann and Peter Beyer
J. Biol. Chem. 2011, 286:8666-8676.
doi: 10.1074/jbc.M110.208017 originally published online January 5, 2011
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 10, pp. 8666–8676, March 11, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Plant Carotene Cis-Trans Isomerase CRTISO
A NEW MEMBER OF THE FADRED-DEPENDENT FLAVOPROTEINS CATALYZING NON-REDOX
□
S
REACTIONS*
Received for publication, December 1, 2010, and in revised form, January 5, 2011 Published, JBC Papers in Press, January 5, 2011, DOI 10.1074/jbc.M110.208017
Qiuju Yu‡, Sandro Ghisla§, Joseph Hirschberg¶, Varda Mann¶, and Peter Beyer‡1
From the ‡Faculty of Biology, Centre for Biological Signaling Studies (Bioss), University of Freiburg, D-79104 Freiburg, Germany, the
§Department of Biology, University of Konstanz, D-78457 Konstanz, Germany and the ¶Department of Genetics, the Alexander
Silberman Life Science Institute, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
The carotene cis-trans isomerase CRTISO is a constituent of
the carotene desaturation pathway as evolved in cyanobacteria
and prevailing in plants, in which a tetra-cis-lycopene species,
termed prolycopene, is formed. CRTISO, an evolutionary
descendant of the bacterial carotene desaturase CRTI, catalyzes
the cis-to-trans isomerization reactions leading to all-trans-ly-
copene, the substrate for the subsequent lycopene cyclization to
form all-trans-␣/␤-carotene. CRTISO and CRTI share a dinu-
cleotide binding motif at the N terminus. Here we report that
this site is occupied by FAD in CRTISO. The reduced form of
this cofactor catalyzes a reaction not involving net redox
changes. Results obtained with C(1)- and C(5)-deaza-FAD sug-
gest mechanistic similarities with type II isopentenyl diphos-
phate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO,
together with lycopene cyclase CRTY and IDI-2, thus represents
the third enzyme in isoprenoid metabolism belonging to the class
of non-redox enzymes depending on reduced flavin for activity.
The regional specificity and the kinetics of the isomerization reac-
tion were investigated in vitro using purified enzyme and biphasic
liposome-based systems carrying specific cis-configured lycopene
species as substrates. The reaction proceeded from cis to trans, rec-
ognizing half-sides of the symmetrical prolycopene and was
accompanied by one trans-to-cis isomerization step specific for the
C(5)-C(6) double bond. Rice lycopene ␤-cyclase (OsLCY-b), when
additionally introduced into the biphasic in vitro system used, was
found to be stereospecific for all-trans-lycopene and allowed the
CRTISO reaction to proceed toward completion by modifying the
thermodynamics of the overall reaction.
tion, carotenoids containing at least one unsubstituted ␤-io-
none ring serve as provitamin A carotenoids (for review, see
Ref. 4). In recent years, a wealth of molecular information on
carotenogenesis in plants and microorganisms has become
available (see Refs. 5, 6 for reviews on plant carotenogenesis),
however, information on the enzymology and biochemistry of
the involved reactions is comparatively scarce.
In the carotenoid biosynthetic pathway, phytoene is the first
carotene formed. Phytoene is a colorless C40 triene prenyl
hydrocarbon, which undergoes four desaturation steps during
which double bonds are introduced to finally form the fully
conjugated undecaene chromophore of the red-colored lyco-
pene (Fig. 1). There are two divergent classes of carotene
desaturases catalyzing the formation of the identical product
(all-trans-lycopene) from the same precursor (15-cis-phy-
toene). The CRTI-type carotene desaturases present in archaea,
bacteria, and fungi are capable of catalyzing the entire reaction
sequence toward trans-lycopene (and beyond in some cases,
forming di- and tetra-dehydrolycopene), additionally mediat-
ing a single cis-to-trans isomerization step at the central C15-
C15Ј double bond. Although the reaction mechanism of CRTI-
type desaturases has not been resolved, all currently available
evidence suggests that no additional protein components are
required. This is contrasted by the complex carotene desatura-
tion system in cyanobacteria and plants, where at least four
gene products are required to achieve the formation of all-
trans-lycopene (Fig. 1). In plants, these are the two carotene
desaturases named phytoene desaturase (PDS)2 and ␨-carotene
desaturase (ZDS) which introduce two double bonds, each.
The PDS and ZDS polypeptides share 27% amino acid sequence
identity and 41% similarity but both are unrelated to CRTI (7,
8). In addition, PDS is a constituent of more or less extended
redox chain and requires additional protein and non-protein
components (9 - 12). The plant desaturase system is comple-
mented by two cis-trans isomerases, represented by Z-ISO (13)
and the carotene cis-trans isomerase CRTISO, which is the sub-
ject of this work. CRTISO isomerizes all cis double bonds
formed by the action of PDS and ZDS (14) yielding 7,9,9Ј,7Ј-
tetra-cis-lycopene3 (commonly referred to as prolycopene; 15)
Carotenoids belong to a large isoprenoid family, some mem-
bers of which are indispensible in all photosynthetic organisms;
however, they can also be formed by some heterotrophic bac-
teria and fungi. In photosynthesis, carotenoids play an impor-
tant role in light harvesting and in the protection of cells from
photo damage caused by reactive oxygen species. In addition,
some carotenoids are precursors of phytohormones such as
abscisic acid (1) and the strigolactones (2, 3). In human nutri-
* This work was supported by The German-Israel Foundation and by the Excel-
lence Initiative of the German Federal and State Governments (EXC 294).
2 The abbreviations used are: PDS, phytoene desaturase; ZDS, ␨-carotene
desaturase; HOMO, highest occupied molecular orbital; IMAC, immobi-
lized metal affinity chromatography; LFER, linear free energy relationship;
SRM, single reaction monitoring.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S8.
1 To whom correspondence should be addressed: Faculty of Biology, Univer-
sity of Freiburg, D-79104 Freiburg, Germany. Fax: 49-761-203-2675; E-mail: 3 The C-atom numbering of carotenes is given according to IUPAC nomencla-
peter.beyer@biologie.uni-freiburg.de.
ture rules for carotenoids.
8666 JOURNAL OF BIOLOGICAL CHEMISTRY
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
ence of respiratory chain substrates indicating a requirement
for reducing conditions (19).
Exactly such conditions were recently found to be required
for lycopene cyclization to proceed. This conversion, as cata-
lyzed by the bacterial CRTY, can also be viewed as a carotene
isomerization reaction. Therefore, the lines of thinking devel-
oped for CRTY might also apply for CRTISO: It was shown that
lycopene cyclization depended on reduced FAD involved in the
stabilization of a transition state. A specific glutamate was also
needed that is believed to be involved in acid/base catalysis (20,
21). Based on this, it was suggested that the sole function of
respiratory chain activities in vitro was to ensure reducing con-
ditions (anaerobiosis), i.e. to prevent (re)oxidation of FADred
(20).
Such evident similarities in the reaction conditions required
by both, CRTISO and CRTY, prompted the research on
CRTISO presented in this report. We show that this enzyme is
FADred-dependent and give details on the regional specificity
and the kinetic properties of the cis-trans isomerization
reaction.
EXPERIMENTAL PROCEDURES
Chemicals Used—Flavin cofactors modified at various posi-
FIGURE 1. Carotene desaturation and isomerization reactions. The reac-
tions have been detailed in a compiled report (22). It contains
references to sources or synthetic methods and to methods for
conversion of riboflavin analogs into the corresponding FAD
derivatives as well as their redox potentials. Flavin cofactor ana-
logs were purified by HPLC, and their identity was verified by
tion sequence starts with 15-cis-phytoene. With bacterial CRTI, a direct con-
version into all-trans-lycopene occurs. This involves four desaturation reac-
tions corresponding to 8 eϪ equivalents (right hand side) and one
isomerization reaction at the C15-C15Ј double bond. In the case of cyanobac-
teria and plants, PDS carries out 2 desaturation steps (4 eϪ) to generate the
9,15,9Ј-tri-cis-␨-caroteneshown. Z-ISOthencatalyzestheisomerizationofthe
centraldoublebondtoform9,9Ј-di-cis-␨-carotene. Subsequently, thedesatu-
rase (ZDS), forms two cis double bonds at the 7 and 7Ј positions (4 eϪ) to yield
7,9,9Ј,7Ј-tetra-cis-lycopene. CRTISO isomerizes 7,9,9Ј-tri-cis-neurosporene
(proneurosporene, not shown) and 7,9,9Ј,7Ј- tetra-cis-lycopene (prolyco-
pene) into the all-trans form. LCY-b/e: lycopene beta/epsilon-cyclase; CRTY:
bacterial lycopene beta-cyclase. CRTISO and CRTI are given in bold to indicate
their homology.
UV/Vis spectroscopy and LC-MS as described (20). Ni2ϩ
-
NTA-agarose resin for protein purification was from Qiagen.
2H2O was obtained from Euriso-top. 5-cis-lycopene, 5,5Јdi-
cis-lycopene and 9-cis-␤-carotene were from CaroteNature
(Switzerland); all-trans-␥-carotene, was a gift from BASF
(Germany). Prolycopene (7,9,9Ј,7Ј-tetra-cis-lycopene) was
extracted with acetone from fruits of the tangerine mutant of
tomato, being defective in CRTISO (16). All other fine chem-
icals employed were purchased from Sigma-Aldrich.
to finally form all-trans-lycopene. The latter is a substrate for
the subsequent introduction of cyclic ␤- and/or ⑀-ionone end
groups to form ␣- and ␤-carotene through the catalysis of the
respective lycopene cyclases. It is interesting to note that
CRTISO shares sequence homology with CRTI, as discussed
above. Hence, it appears plausible that CRTISO originated
from CRTI (7, 16, 17).
Construction of the CRTISO Expression Vector—The cDNA
of CRTISO from tomato (16), from which the first 79 codons of
the transit peptide were truncated, was inserted into the BamHI
site behind a His6 tag, an 83 amino acids lipoyl domain and a
TEV protease cleavage site (HLT domain) (23) in the plasmid
vector pHis-Parallel2 (24). The resulting plasmid, termed
pHLT-CRTISO, was transfected into E. coli cells of the strain
BL21(DE3) harboring the plasmid pGro7 (Takara), which
encodes the groES-groEL-chaperone system under the control
of an arabinose-inducible promoter.
One feature of CRTISO, which it shares with carotene
desaturases of both types, is the presence of a GXGXXG-bear-
ing dinucleotide-binding motif in proximity of the N terminus
of the polypeptide (18). Whereas the presence of a redox-active
cofactor is a necessity in desaturases catalyzing net electron
transfer, such a requirement is not evident for the cis-trans
isomerase CRTISO, because the reaction it catalyzes does not
involve redox changes. A cryptic electron transfer might be
envisaged to occur transiently. However, in the absence of
information on the nature of the cofactor mechanistic consid-
erations are just speculative. Earlier attempts to shed light on
the reaction mechanism showed CRTISO to be dependent on
dialyzable cytoplasmatic components as well as on plasma
membranes of Escherichia coli. Furthermore, CRTISO was
shown to depend on the redox state of the respiratory chain. In
Expression and Purification of CRTISO—Bacteria trans-
formed with pHLT-CRTISO were grown in 2*YT-medium
under continuous shaking at 37 °C till an A600 of 0.6 and then
induced by adding arabinose (8 mM) and IPTG (0.2 mM). The
induction took place over night at 16 °C. Cultures were har-
vested and frozen at Ϫ20 °C for further use.
Protein purification was carried out on ice. Cell pellets were
resuspended in buffer A (25 mM phosphate-sodium buffer, pH
6.2; MgCl2, 2.5 mM; NaCl, 300 mM; glycerol 15%, 1.5 ml buffer
A/g cell mass) and disintegrated by two passages through a
particular, the isomerization reaction accelerated in the pres- French Press Cell, operated at 18,000 psi. After the removal of
MARCH 11, 2011•VOLUME 286•NUMBER 10
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
cell debris by centrifugation at 17,000 ϫ g for 15 min, the super- pene was also used for the “unknown” peak (e) since they share
natant was solubilized with Tween 20 at a final concentration of similar spectra with a trans-like fine structure (25).
10ϫ CMC (0.067%) for 30 min. The solubilized protein was
The standard CRTISO assay mixture (final volume 200 ␮l)
then allowed to bind Ni2ϩ-NTA-agarose (Qiagen) equilibrated consisted of 163 ␮l of buffer A which was supplemented with
with buffer A containing 0.067% Tween 20. The binding step typically 30 ␮l of the substrate-containing liposome suspension
was performed for 45 min under shaking at 37 rpm and fol- to result in a final carotene concentration of 5 ␮M. The final
lowed by three washing steps with buffer A containing 0.02% concentration of FAD was 50 ␮M. CRTISO was added at a 1 ␮M
Tween 20 and 4 mM imidazole. Elution of the bound protein final concentration typically added to the assay in 5 ␮l, leading
was accomplished with buffer A containing 100 mM imidazole to a final Tween 20 concentration of ϳ0.0005% (1ϫ CMC ϭ
and 0.02% Tween 20. The preparation was used to record the 0.0067%). Reducing conditions were obtained by supplement-
UV/Vis spectra (Shimadzu, UV-2501PC) or dialyzed against ing the assay with 2 ␮l of freshly prepared 0.1 M Na-dithionite
buffer A to remove imidazole before freezing aliquots at solution. All solutions were equilibrated with N2 before use and
Ϫ80 °C.
the reactions carried out in a glove box under an N2 atmosphere
Protein concentrations were determined with the Bradford in very dim daylight at 37 °C for 15 min, unless otherwise indi-
method. Protein purity was checked by SDS-PAGE using 10% cated. Time course experiments were performed under stand-
polyacrylamide gels. Proteins were detected using Coomassie ard conditions. The reactions were terminated at various inter-
Brilliant Blue G250 (Sigma-Aldrich). For the identification of vals by mixing with an equal volume of chloroform/methanol
protein-bound cofactors, CRTISO was purified essentially fol- 2:1 (v/v).
lowing the procedures described above, however, glycerol and
Cyclization reactions of carotenes were carried out using
Tween 20 were omitted from the elution buffer. 10 mg of such lycopene ␤-cyclase from Oryza sativa (OsLCY-b). Because the
protein was heat-denatured the protein removed by centrifuga- reaction conditions of OsLCY-b are essentially identical with
tion. The supernatant was lyophilized and the residue dissolved those of CRTY including anaerobosis (20), 5 ␮g of IMAC-puri-
in 50 ␮l of water of which 4 ␮l were used for LC-MS analysis.
fied OsLCY-b protein was added to the standard CRTISO
Apo-CRTISO Preparation and Reconstitution—The CRTISO assays.
apo-enzyme was prepared according to published procedures
For deuteration experiments, all buffers, the Na-dithionite
(20) with some modifications. IMAC- purified protein (2 and FAD stock solutions as well as the lycopene-liposome sus-
mg/ml) was dialyzed for 16 h at 4 °C against buffer A containing pension were prepared, as described above, but in 2H2O. The
2 M KBr. The KBr was removed by dialysis against buffer A and protein, added in very small volume (around 2.5 vol %) was in
eventually occurring precipitates were removed by centrifuga- H2O.
tion at 21,000 ϫ g for 15 min. Absence of cofactors was con-
Extraction and Analytical Methods—Enzymatic assays were
firmed by UV/Vis spectroscopy, fluorescence measurement, extracted twice with chloroform/methanol 2:1 (v/v). The
and by the absence of enzymatic activity. organic phases containing substrate and products were com-
Cofactor reconstitution of the apo-protein was carried out bined and dried under reduced pressure. The residue was dis-
under a N2 atmosphere. 100 ␮l of 200 ␮M flavin analogs were solved in chloroform and analyzed by HPLC-System 1 (Waters,
first reduced with 4 ␮l of freshly prepared 0.1 M Na-dithionite Alliance 2695) on a C30 reversed phase column (YMC-Europe)
solution of which 50-␮l aliquots were then transferred to 115 ␮l as detailed earlier (20) or dissolved in hexane and analyzed by
of buffer A to which 200 pmol of apo-enzyme was added HPLC-System 2 (UFLC, Shimadzu, Prominence) using a direct
(around 5 ␮l, MWCRTISO ϭ 67 kDa). The mixtures were incu- phase column (Nucleosil 300–5, Macherey & Nagel) with dry
bated at room temperature for 1 h; then the reaction was initi- hexane/N-ethyldiisopropylamine 2000:1 (v/v) as the mobile
ated by adding 30 ␮l of prolycopene liposomes which led to the phase at a flow-rate of 1.5 ml/min (25). Equilibration of this
final prolycopene concentration of 5 ␮M. Enzyme kinetic stud- direct phase system can take several days. Both HPLC systems
ies to determine the Km of FAD were performed analogously were equipped with a photodiode array detector (PDA).
using an incubation time of 15 min. The final concentrations of
NADP(H), NAD(H), FAD, FMN, and flavin analogs were
analyzed and purified using HPLC-System 3, and identified by
FAD were 0.2, 0.25, 0.4, 1, 2, 5, and 10 ␮M.
Enzymatic Assays—Protein-free liposomes containing caro- LC-MS-MS using ESI ionization, as previously described (20).
tene substrates were prepared with soybean lecithin (Sigma-
Prolycopene was extracted from the “Tangerine” variety of
Aldrich) in buffer A according to previously published methods tomato fruit with acetone. After partition against petroleum
(20). The incorporation of carotenes into the lipid bilayer was ether/diethyl ether 2:1 (v/v), the organic phase was dried. After
assessed by extracting the carotenes from the liposomes formed dissolving in CHCl3, the extract was applied to a self-packed
with chloroform/methanol 2:1 (v/v) followed by spectrophoto- silica gel column, which was developed with n-hexane to isolate
metric estimation (Shimadzu, UV-2501PC) using ⑀470
ϭ
ϭ
ϭ
the hydrocarbon fraction. Prolycopene was then purified on a 3
␮m C18 RP column (Hypersil Gold, Thermo-Fisher Scientific)
using acetonitrile isocratically at a flow-rate of 1.4 ml/min
nm
Ϫ1
Ϫ1
187,000 liters mol cm for all-trans-lycopene, ⑀470 nm
Ϫ1
Ϫ1
184,000 liters mol cm for 5-cis- lycopene and ⑀439 nm
105,000 liters molϪ1 cmϪ1 for prolycopene. Additional molar (HPLC-System 4).
Ϫ1
extinction coefficients used were ⑀468 nm ϭ 154,000 liters mol
For LC-MS of carotenes in the deuteration experiment we
cmϪ1 for 7-cis- lycopene, ⑀465 nm ϭ 169,000 liters molϪ1 cm
utilized HPLC-System 5, as described earlier (20) coupled to a
Ϫ1
Ϫ1
Ϫ1
for 9-cis- lycopene, ⑀444 nm ϭ 115,000 liters mol cm for Thermo-Fisher Scientific LTQ instrument. Carotenes were
7,9-di-cis-lycopene. The extinction coefficient for 9-cis-lyco- separated on a C30 RP (Thermo-Fisher Scientific) column,
8668 JOURNAL OF BIOLOGICAL CHEMISTRY
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
APCI-ionized using N2 as reagent gas and analyzed in the pos-
itive ion mode. Separated carotenes were identified by their
spectra, retention times in comparison with authentic refer-
ences and by their quasi-molecular (Mϩ1) ions.
Procedures Used for Kinetic Simulations—The time depend-
ence of concentrations of lycopene species (a)–(g) was simu-
lated using the global analysis program Specfitꢀ, which solves
the series of differential equations representing the reaction
sequences shown in Fig. 8. Because the conditions of catalysis in
the membrane system used cannot be defined, the simplifying
assumption was made that the interaction constants for
enzyme and the various lycopene species are similar. Conse-
quently, steps that lead to the formation of the corresponding
Michaelis-Menten complexes were not included in the simula-
tion. The simulation/fitting procedure consists in a gradual
approach in which values for the various steps are first fixed in
a range compatible with the measured kobs values; the system
then fits the experimental data representing the disappearance
of species (a) in a first step. Subsequently, the rates of steps that
affect the time dependent concentrations of single, specific spe-
cies ((a)–(g) are optimized while the others are held “fixed”).
Finally a fit of the data (curves) obtained from the simulation
and of the experimental data points is done with the application
KaleidaGraph using the same mono- or bi-exponential equa-
tion (of the type: y ϭ AꢁeϪk1t ϩ BꢁeϪk2t ϩ C, where A and B are
amplitudes and C the initial value). For more detailed informa-
tion, refer to Gradinaru et al. (26).
RESULTS
CRTISO Requires Anaerobic Conditions for Activity—The
carotene cis-trans isomerase CRTISO was cloned from tomato
(16) and expressed in E. coli in the form of a N-terminal His6
fusion. Differential centrifugation and IMAC produced a near
homogeneous protein preparation which was used in the inves-
tigations (supplemental Fig. S1). Inspired by results obtained
with two other isomerases involved in isoprenoid metabolism,
namely type II isopentenyl diphosphate: dimethylallyl diphos-
phate isomerase (IDI-2; see Ref. 27 and citations therein) and
FIGURE 2. In vitro assay of CRTISO isomerization reaction with 7,9,9ꢂ,7ꢂ-
tetra-cis-lycopene (prolycopene) as a substrate. A, CRTISO isomerizes yel-
low prolycopene into pink-colored products, shown in the liposome-contain-
ing assay (left) and after carotenoid extraction in organic solution (right). A
30-min assay (standard conditions) is shown. Con, control incubation in the
absence of CRTISO. B, UV/Vis spectra (in hexane) of the extracted assays
shown above. Showing the bathochromic shift of the spectra. C, HPLC sepa-
ration of the assays shown above on 3 ␮m C30 RP column (HPLC-system 1).
Prolycopene (peak (a)) was isomerized into mainly all-trans-lycopene (peak
(g)) by CRTISO.
lycopene ␤-cyclase (CRTY/LCY-b; 20, 21) both catalyzing centrated protein sample shown in Fig. 3D (left panel) is com-
FADred-dependent non-redox reactions, we hypothesized a patible with the presence of a flavin, FAD or FMN in the oxi-
similar mechanism operating with CRTISO. In fact, under a dized state. Gel filtration removed bound cofactor beyond the
nitrogen atmosphere used to maintain FADred, the 7,9,9Ј,7Ј- detection limit. To reveal its nature, the IMAC-purified protein
tetra-cis-lycopene (prolycopene) substrate present in the assays was heat-denatured and the resulting soluble fraction subjected
embedded into phosphatidylcholine liposomal membranes was to HPLC-MS-MS analysis, optimized to unequivocally identify
readily converted into mainly all-trans-lycopene at a high dinucleotide cofactors based on retention time, UV/Vis spec-
velocity. The reaction can be monitored visually by the ϳ40 nm tra, molecular ions, and Single Reaction Ion Monitoring
bathochromic shift of the UV/Vis spectrum upon cis-to-trans employing the specific MS2 daughter ions as the diagnostic tool
isomerization of prolycopene (Fig. 2, A and B). This reflects the (Fig. 3, A–C). Based on retention time and UV/Vis spectrum,
enhanced electron delocalization in the system consisting of FAD was the only detectable cofactor, while mass spectrometry
eleven double bonds (see structure in Fig. 1). Analysis of the revealed the additional presence of some FMN and NAD, how-
reaction on a C30 RP column revealed all-trans-lycopene as the ever too small in amount to be photometrically detectable.
main lycopene species formed, accompanied by certain
The nature of the effective cofactor, being either FAD or
amounts of newly formed cis isomers (Fig. 2C). CRTISO activity FMN, was further assessed with enzymatic assays. Both, IMAC-
did not exhibit a pH dependence in the range of 5.8–8.0.
purified protein and apo-CRTISO were incubated in a N2
CRTISO Catalysis Requires Reduced FAD (FADred)—The atmosphere with either of the cofactors and in the presence/
IMAC-eluted protein had a very faint yellow color, suggesting absence of Na-dithionite (Fig. 4A). Whereas oxidized FAD (in
the presence of some small amounts of protein-bound flavin, the absence of the reductant) was ineffective (I), FADred was
i.e. of holo-protein. The UV/Vis spectrum obtained with a con- able to drive the reaction at a rate of 3.6 pmol min ␮gϪ1 (II).
Ϫ1
MARCH 11, 2011•VOLUME 286•NUMBER 10
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
reactivation of the apo-protein was obtained in the absence of
ϩ
cofactors (VI) and in the presence of NADH H as the sole
cofactor (VII). Moreover, IMAC-purified protein exhibited
only little activity in the presence of reductant when free FAD
was not added (X). FMNred was able to substitute for FADred
but was about 60% less effective (IX). As with FAD, reduction of
FMN was possible in the presence of high concentrations of
ϩ
NADH H (VIII). In conclusion, FADred appears to be the
cofactor of choice while FMNred is only partially active. Under
the conditions detailed under “Experimental Procedures,” the
K
m for FADred was Ϸ 0.55 ␮M (Fig. 4B).
Flavin Analogs Used as Mechanistic Probes—Flavin analogs
are instrumental for the study of mechanism of flavin-depen-
dent catalysis. In the present work we used C(5)-deaza- and
Ϫ
Ϫ
C(1)-deaza-FAD to discriminate between 1e and 2e redox
chemistry (28). Using C(1)-deaza-FADred, a conversion rate of 3
pmol min ␮gϪ1 was found while the system was fully inactive
Ϫ1
with C(5)-deaza-FADred (Fig. 4C).
A series of FAD analogs substituted at position C(8) was then
used to assess a possible dependence of the conversion rate on
the flavin redox potential (Linear Free Energy Relationship,
LFER, Ref. 29) in analogy to the study carried out recently with
bacterial lycopene cyclase CRTY (20). With C(8)-O-CH3, C(8)-
CH3 (FAD), C(8)-Cl, and C(8)-SO2-CH3 (in decreasing order of
their midpoint potential; see supplemental Fig. S2 for struc-
tures) conversion rates of 2.94 Ϯ 0.23, 3.62 Ϯ 0.31, 4.16 Ϯ 0.28,
Ϫ1
and 0.3 Ϯ 0.08 pmol min ␮gϪ1 (mean Ϯ S.E.), respectively,
were found. Thus, in contrast to the case of CRTY, a LFER
relationship was not found (see “Discussion”).
The mechanism of isomerization reactions relying on acid-
base catalysis can be identified by carrying out the reaction in
labeled solvent, when the sites of proton attack and abstraction
are not identical. The lycopene cyclase CRTY is an example (see
Ref. 20 and citations therein). Prolycopene isomerization
experiments were therefore carried out in 2H2O otherwise
maintaining the standard assay conditions. Analysis of the reac-
tion products by LC-MS showed the absence of mass gain, i.e. of
deuterium incorporation (supplemental Fig. S3).
Characterization of the Catalytic cis-to-trans Isomerization
Sequences—Assigning regional specificities of cis-trans isomer-
ization is not trivial with lycopene isolated in small amounts
from biological samples because NMR is the only applicable
methodology to elucidate the resulting structures. Only few
lycopene cis isomers possess unique UV/Vis spectral features
such as prolycopene (Fig. 2B) or central cis isomers such as
FIGURE 3. Identification of cofactors bound to CRTISO. 10 mg of IMAC-
purified CRTISO was heat-denatured. After centrifugation and concentration,
4 ␮l of supernatant was applied to LC (system 3)-MS-MS. A, the UV/VIS trace
recorded at 450 nm and the corresponding spectrum of peak2 (D, right panel)
demonstrates flavin presence. B, SRM, using the MS2 daughter ions (in brack-
ets) previously identified with cofactor standards (shown in C) was employed
to detect the presence of NADϩ (peak 1), FAD (peak 2), and FMN (peak 3).
NADϩ was present in trace amounts only since it was undetectable in the UV.
FMN was present in trace amounts as seen in the accompanying UV/Vis trace
(A). No MS-SRM signal was observed for NADPϩ (trace not shown). D, left: 13-cis or 15-cis-lycopene exhibiting a strong “cis-peak” at 360
UV/Vis absorption spectrum of IMAC-purified and concentrated CRTISO.
nm. We therefore resorted to work published by Hengartner et
al. (25) describing the chemical synthesis and NMR-spectro-
ϩ
NADH H , at high (2 mM) concentrations was able to partially scopic characterization of a series of specific lycopene cis iso-
substitute for Na-dithionite as an FAD reductant (III), but did mers, some of which represent members of the eight possible
not support catalysis as the sole cofactor (IV). In our interpre- intermediates existing between prolycopene (7,9,9Ј,7Ј-tetra-
tation, this residual activity is due to an NADH dependent cis-lycopene (a); Fig. 8) and all-trans-lycopene (g), the species
reduction of a small fraction of the enzyme that contains FAD with the lowest ground state of energy. Where applicable, the
(holo-protein). In agreement with this, IMAC-purified authors provided to us the corresponding unpublished original
CRTISO that was subjected to KBr-treatment to remove all UV spectra of this work (supplemental Fig. S4). In addition, we
FAD (apo-protein) had no activity. Subsequent addition of adopted the direct phase HPLC system they used (HPLC-Sys-
reduced FAD led to CRTISO reconstitution (V) as shown by tem 2) which allowed structural assignments by comparison of
specific conversion rates similar to those obtained in (II). No the elution order and time normalized relative to the elution
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
time of all-trans-lycopene. Using this HPLC system, five iso-
mers were resolved which were formed enzymatically at the
expense of prolycopene (peak (a) in Fig. 5, A and B). The main
product formed was all-trans-lycopene (peak (g)) accompanied
by smaller amounts of 5-cis-lycopene (peak (f), both identified
by internal standardization with authentic references). Follow-
ing the logic of an ordered cis-to-trans isomerization reaction,
the formation of 5-cis-lycopene (f) is somewhat unexpected
since this double bond is trans-configured in prolycopene. Peak
(b) unequivocally, represents, 7,9-di-cis-lycopene, in which
only one half-side of the symmetrical prolycopene molecule is
isomerized to trans. The C(7) and C(9) mono-cis species (c, d)
eluted at their expected position and showed absorption max-
ima as well as spectral fine structures that matched the refer-
ence spectra (for UV/Vis reference spectra and spectra
observed see supplemental Figs. S4 and S5, respectively). One
additional lycopene species (e) was enzymatically formed but
could not be structurally assigned.
It is relevant to note that some of the expected lycopene
cis-species were not formed (supplemental Fig. S6). These were
the symmetrical di-cis-lycopenes (9,9Ј-di-cis-lycopene and
7,7Ј-di-cis-lycopene). Similarly, the identified 5-cis-lycopene (f)
was not accompanied by the respective di-cis species (arrows in
Fig. 5A indicate their expected elution). In addition, there was
no indication for the occurrence of tri-cis-lycopenes, i.e. 7,9,9Ј-
tri-cis-lycopene and 7,7Ј9Ј-tri-cis-lycopene.
Kinetic Course of Chemical Transformations Catalyzed by
CRTISO—The time courses of the formation and disappear-
ance of species occurring upon incubation of 7,9,9Ј,7Ј-tetra-cis-
lycopene with CRTISO was analyzed as shown in Fig. 5, C and
D. The disappearance of prolycopene (a), the appearances of
the intermediates (e, f) and of the final product all-trans-lyco-
pene (g) were analyzed using a monoexponential fitting routine;
the appearance and decay of the intermediate species (b), (c),
and (d) were analyzed using a bi-exponential fit. The obtained
apparent rates (k1-obs) are listed in Table 1. They reflect an
approach to equilibria and thus reflect all the intrinsic rates of
formation and decay of a given species (30). In accordance with
this, the equilibrium situation was disrupted experimentally by
the addition of lycopene cyclase from rice OsLCY-b (20), an
enzyme that converts all-trans-lycopene (g) essentially irre-
versibly into ␤-carotene (h; Fig. 6), This is evidenced by the
rapid disappearance of all lycopene species (a)-(g). In an
attempt to verify this interpretation we simulated the time
course of concentration changes of relevant species in a system
assumed to represent the experimental set-up described in Fig.
CRTISO; II. 100 ␮M FAD ϩ Na-dithionite ϩ IMAC-purified CRTISO; III. 100 ␮M
FAD ϩ 2 mM NADH ϩ IMAC-purified CRTISO; IV. 2 mM NADH ϩ IMAC-purified
CRTISO; V. 100 ␮M FAD ϩ dithionite ϩ apo-CRTISO; VI. apo-CRTISO; VII. 2 mM
NADH ϩ apo-CRISO; VIII. 100 ␮M FMN ϩ 2 mM NADH ϩ IMAC-purified CRTISO;
IX. 100 ␮M FMN ϩ dithionite ϩ IMAC-purified CRTISO; X. IMAC-purified
CRTISO ϩ dithionite; XI. Control, no CRTISO and cofactors added. B, Lin-
eweaver-Burk plot for FAD (0.2, 0.25, 0.4, 1, 2, 5, 10 ␮M) carried out with apo-
CRTISO. Reflavinylation of CRTISO was allowed to take place at room temper-
ature for 1 h (y ϭ 0.15x ϩ 0.27; Km Ϸ555 nM). C, reconstitution of apo-CRTISO
FIGURE 4. A, cofactor requirement of CRTISO. Enzymatic assays were carried with deaza-flavins. The assays were performed according to the standard
out under an N2 atmosphere with 200 pmol of purified CRTISO (E) and proly- procedures given under “Experimental Procedures.” 1. apo-CRTISO; 2. apo-
copene liposome (final concentration of prolycopene, 5 ␮M) in 200 ␮l. The CRTISO ϩ FADred; 3. apo-CRTISO ϩ C(1)-deaza-FADred; 4. apo-CRTISO ϩ C(5)-
assays were incubated for 15 min at 37 °C. I. 100 ␮M FAD ϩ IMAC-purified deaza-FADred. Lyc.: lycopene, DTN: Na-dithionite.
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
losing coincidence with the experimental data. This suggests a
good approximation to the intrinsic values. Only narrow varia-
tion is also allowed with respect to the sum of the rates of steps
leading to (g) or (f). In contrast, the time course simulation for
the formation of all-trans-lycopene allows considerable varia-
tion, provided the sum of k-values remains constant. This is due
to the presence of four parallel single pathways originating from
(c), (d), (e), and (f) leading to all-trans-lycopene (g),. We
selected the values for k12 to k16 to best fit the “equilibrium
abundances” (plateaus) of species (c), (d), and (e).
The ratios of “forward” and “reverse” steps (see legend of Fig.
8) are in agreement with the presence of equilibria. For species
(a) and (b) the ratio is 2, while for the transformation of (b) into
(c), (d), and (e) the equilibrium appears to be in favor of (b). This
contrasts with the equilibria linking species (c), (d), and (e) to (f)
and (g), where ratios of 40–60 in favor of (g) are found. A high
ratio of Ϸ100 for k17/k18 is necessary for simulating the
observed plateau concentration of species (f). These latter high
ratios might be related to the experimental difficulty observed
in driving the isomerization reaction “backwards” starting from
all-trans-lycopene ((g), see supplemental Fig. S8). It is also
noted that the abundance of the various lycopene species at
equilibrium corresponds roughly to their relative theoretical
energy levels calculated by Guo et al. (31).
The simulations were validated by fitting both the experi-
mental and the simulation data using the same algorithms. A
comparison of the results listed in Table 1 shows that in the case
of the most abundant lycopene species (a), (b), (f), and (g) the
coincidence of kobs for both sets of data is good. With the spe-
cies (c), (d), and (e), the abundance of which was Յ 2%, the
scatter of experimental data were large and the match corre-
spondingly less pronounced.
FIGURE 5. Time course of the CRTISO reaction and identification of inter-
mediates. A, separation of lycopene isomers formed in a 15-min CRTISO
standardreaction(directphaseHPLC-system2). Arrowsindicatetheexpected
position of the di-cis isomers given (as explained in the text). Asterisks denote
cis isomers that were artifactually formed. Letters denote enzymatic products
as given in Fig. 8. (a), 7,9,9Ј,7Ј-tetra-cis-lycopene (prolycopene); (b) 7,9-di-cis-
lycopene; (c) 9-cis-lycopene; (d) 7-cis-lycopene; (e) unidentified cis-lycopene;
(f) 5-cis-lycopene; (g) all-trans-lycopene. See supplemental Fig. S5 for UV/VIS
spectra. B, chromatographic runs of a time course (HPLC-system 2). C and D,
time courses of species involved in prolycopene isomerization under stand-
The results of the simulations provide arguments to assess
the possible sequences and courses of isomerizations depicted
in Fig. 8. Several alternative pathways are represented, which
can all lead to the final product all-trans-lycopene (g). Steps
denoted with (?) link the species in (c), (d), and (e) in the middle
row. Thus formation of (g) from (b) might be envisaged to pass
in sequence e.g. via (e), (c), and (d). However, the results of the
ard assay conditions. C, disappearance of the substrate and formation of the simulations suggest the absence of rapid steps interlinking (c),
final product; D, time courses of intermediate lycopene species (for structures
see Fig. 8); note the different scales of the y-axes. The symbols denote exper-
imental data points and are the average of two independent measurements,
bars represent data scatter. The lines through the data points are fits. E, sym-
(d), and (e). Such rapid steps would lead to similar (observed)
rates of formation for these species, which is not observed (Fig.
5D, Table 1). Fig. 8 represents a minimal scheme, it does not
bols and color codes are as in C and D. The lines through the data points are
exclude the occurrence of further species and combinations of
the results of the simulations described in the text. Data points for (c) and (e)
steps.
have very low values between 0 and 2%, the corresponding profiles are sim-
ilar to those shown in Fig. 5D; they are not shown for clarity. See Table 1 for
Lycopene cis Isomers in Lycopene Cyclization—The introduc-
kobs values.
tion of lycopene cyclase OsLCY-b into the CRTISO-containing
system, as discussed above (Fig. 6) led to the formation of all-
5, C and D. The simulation was based on the conversion scheme trans-␤-carotene. When 5-cis-lycopene was used as a substrate,
depicted in Fig. 8 and includes all species shown and the steps k1 with CRTISO plus OsLCY-b the monocyclic ␥-carotene
to k18 with their rate constants. As shown in Fig. 5E, there is formed, in addition to all-trans-␤-carotene (Fig. 7B, traces III,
satisfactory coincidence of experimental data points with the IV). This monocyclic carotene was hypothesized to derive from
corresponding simulation curves. Importantly, “plateaus” 5-cis-lycopene (with the 5-cis-end not being amenable to cycli-
reflecting equilibrium concentrations of lycopene species were zation). At longer (30 min) incubation time, this end was
attained at the end of the time axis which were similar to those isomerized to trans by CRTISO, allowing the accumulation of
determined experimentally.
all-trans-␤-carotene (Fig. 7B, trace V). To prove this assump-
In these simulations, the magnitude of steps k1/k2 can only be tion, we incubated 5-cis-lycopene with OsLCY-b (in the
varied within a narrow range (Ϯ 20% of the listed rates) without absence of CRTISO, Fig. 7C, trace VI). This yielded a ␥-caro-
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
TABLE 1
Apparent rate constants for the formation and decay of species in comparison with the simulated model
For structures of lycopene species see Fig. 8. Experimental (Fig. 5, C and D) and simulation (Fig. 5E) data points were fitted using the same algorithms, i.e. a mono-
exponential equation for (a),(e),(f),(g) and a bi-exponential one for (b),(c),(d).
k
1-obs (min؊1) experimental
k
2-obs (min؊1) experimental
Lycopene isomer
Formation/ decay
(simulation)
؎S.E.
(simulation)
؎S.E.
(a) 7,9,9´,7´-tetra-cis
d
0.068
(0.073)
0.4
0.002
(0.001)
0.06
–
(b) 7,9-di-cis
(c) 9-cis
f/d
f/d
f/d
f
0.057
0.01
(0.29)
0.12
(0.002)
0.04
(0.065)
0.014
(0.004)
0.02
(0.021)
0.27
(0.006)
0.09
(0.007)
0.001
(0.002)
0.001
(d) 7-cis
(0.22)
0.11
(0.006)
0.01
(0.07)
(0.002)
a
(e) “unknown”
(f) 5-cis
(0.52)
0.067
(0.042)
0.050
(0.060)
(0.01)
0.003
(0.001)
0.003
(0.001)
a
f
(g) all-trans
f
–
a For species (e), formed at low concentrations, both types of fit could be applied yielding very similar k1-obs values, while the apparent rate of decay k2-obs obtained from a
bi-exponential fit is very low (k1 Ն 100 ϫ k2). For species (f) a bi-exponential fit did not yield meaningful results.
epoxycarotenoid oxygenases (NCEDs). With the suitable in
vitro system at hand, the question arose whether CRTISO in
combination with OsLCY-b can decide on the proportion of
9-cis configured ␤-carotene and derived xanthophylls thus
playing a regulatory role. To test, we isolated 7-cis and 9-cis-
lycopene from isomerization assays (peaks (c) and (d) in Fig.
5A), incorporated them into liposomes to run lycopene cycliza-
tion assays with OsLCY-b. Again, no ␤-carotene was formed;
instead, two monocyclic cis-configured ␥-carotene species
accumulated which are presumed to represent the 7- and
9-mono-cis forms, respectively (Fig. 7D).
DISCUSSION
Role and Redox State of FAD—CRTISO is the cis-trans caro-
tene isomerase acting at the stage of tri-cis-neurosporene and
tetra-cis-lycopene (prolycopene, 19), both representing canon-
FIGURE 6. Addition of lycopene ␤-cyclase disrupts the chemical equilib-
rium in standard CRTISO assays. A, negative control (no enzyme added).
The equilibrium between substrate and products in the CRTISO assay carried
out under standard conditions was established after an incubation time of 60
ical desaturation intermediates in the carotenogenesis of cya-
nobacteria and plants (14). CRTISO is required to drive caro-
tenogenesis in non-green tissues, while there is no strict
min (B, comp. Fig. 5), because it remained unchanged after 90 min of incuba-
requirement in green tissues. For instance, developing leaves of
tion (C). The addition of lycopene cyclase OsLCY-b at the 60th minute led to
the “Tangerine” variety of tomato show bleaching because they
the complete conversion of all lycopenes into mainly all-trans-␤-carotene
(peak h) after additional 30 min of incubation time (D). (a) Prolycopene; (b)
7,9-di-cis-lycopene; (c) 9-cis-lycopene; (d) 7-cis-lycopene; (g) all-trans-lyco-
pene; (f) 5-cis-lycopene.
are defective in CRTISO, but mature leaves are phenotypically
unaffected (16). CRTISO requirement is also pronounced in
developing seedlings, facilitating chloroplast development dur-
tene species of which it appeared reasonable to assume that it ing seedling germination and photomorphogenesis (17). With
carried the 5-cis double bond in the aliphatic chain. This is the photosynthetic apparatus once established, the isomeriza-
because the simultaneous presence of CRTISO was required to tion reaction is thought to be substituted by photo-isomeriza-
achieve a 100% conversion into all-trans-␤-carotene without tion (17, 32, 33), which, however, requires a hitherto unknown
any notable formation of ␥-carotene (Fig. 7C, trace VII). This sensitizer (16).
shows that CRTISO not only acts on the aliphatic lycopene
CRTISO possesses a dinucleotide binding domain and shares
chain (Fig. 7A) but also on monocyclic carotenoids. These this property with the bacterial carotene desaturase CRTI, to
results also exclude for a potential cis-trans isomerization side which it is evolutionarily, related. The nature of the cofactor
activity of OsLCY-b (being itself a FADred-dependent isomer- needed was unknown. Based on our data, FAD is the cofactor
ase; 20).
required by CRTISO; trace amounts of FMN and NAD might
The 5-cis configuration of lycopene may forbid cyclization occur by cofactor exchange in E. coli since FAD is not tightly
because this C(5)-C(6) double bond is mechanistically involved bound. We can state further that it is the reduced form of
in lycopene cyclization (20, 21) while double bonds in a more FAD, partially replaceable by FMNred, which drives enzy-
central position are not. 9-cis-configured double bonds are matic activity of CRTISO. This calls for revisiting the previ-
especially interesting since 9-cis configured xanthophylls rep- ous findings mentioned (see Introduction). Oxygen deple-
resent a molecular “earmark” to direct them into the biosynthe- tion through respiration apparently prevents FADred
sis of abscisic acid (ABA) through the action of 9-mono-cis oxidation in the complex E. coli system used (19). Taken
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
Similarities and Dissimilarities between CRTISO, CRTY, and
IDI-2—There are two additional enzymes involved in isopre-
noid metabolism that belong to this class of FADred-dependent
non-redox enzymes, both representing non-homologous
isomerases, namely isopentenyl-diphosphate isomerase type 2
(IDI-2) and lycopene ␤-cyclase (LCY-b in plants, CRTY in bac-
teria). Modified flavins, in particular C(1)- and C(5)-deaza-fla-
vins have been used in all three cases as probes to unravel pos-
sible reaction mechanisms. CRTISO is similar to IDI-2 (34)
because both were inactive when reconstituted with C(5)-
deaza-flavins, whereas they were active with C(1)-deaza-fla-
vins. This is contrasted by CRTY where “normal” activity was
found with C(5)-deaza-FAD (20). The fact that C(5)-deaza-fla-
Ϫ
vins do not support 1e transfer (28) together with the finding
of an LFER led us to propose a CRTY reaction mechanism not
involving transient (cryptic) electron transfer. In the present
case, the absence of an observable LFER (with flavin analogs
carrying substituents at position C(8)), may be explained by the
occurrence of a rate-limiting step that is not linked to the chem-
ical event.
In view of the similarities of the catalyzed reactions and the
requirement for reduced flavin it would appear reasonable to
assume a similar basic chemical mechanism in all three cases
(IDI-2, CRTY, CRTISO). However, this conflicts with C(5)-
deaza-flavin being active with CRTY but not with CRTISO and
IDI-2. The question therefore arises whether the mechanisms
of these enzymes differ in a fundamental aspect. There are var-
ious possibilities to rationalize the absence of activity of
CRTISO/IDI-2 with C(5)-deaza-flavins. The flavin N(5) posi-
tion could be involved in the formation of a crucial H-bridge or
play an active role in acid/base catalysis (as suggested for IDI-2;
27). Alternatively, in the case of a charge transfer mechanism,
such as suggested for CRTY, it is conceivable that the absence of
the specific N(5) HOMO orbital of reduced flavin in CRTISO/
IDI-2 precludes a productive overlap interaction with the tran-
FIGURE 7. Stereoselectivity of lycopene ␤-cyclase (OsLCY-b). A, CRTISO
can isomerize 5-cis-lycopene (f) into all-trans-lycopene (g), but not in the
reverse direction (see supplemental Fig. S8). (I) Control (incubated in the
absence of CRTISO), (b) upon 30 min of incubation in the presence of CRTISO.
B, this cis-to-trans isomerization process is accelerated when CRTISO and
OsLCY-b are combined. OsLCY-b was added after 30 min of CRTISO reaction
(as in (II)) and the reaction was analyzed after 5 (III), 10 (IV), and 30 min (V).
Monocyclic 5-cis-␥-carotene formed from 5-cis-lycopene and all-trans-␤-car-
otene formed from all-trans-lycopene and/or from all-trans-␥-carotene. This
is inferred from C, where 5-cis-lycopene (f) was incubated with OsLCY-b in the
absence of CRTISO (VI) yielding only 5-cis-␥-carotene, while all-trans-␤-caro-
tene was only formed when CRTISO was added simultaneously (VII). D, 7- and
9-cis isomers were collected (peaks (d) and (c) in Fig. 5). They do not separate
on HPLC-System 1 used here (VIII); (IX) shows the elution of an all-trans-␥-
carotene standard. Upon incubation with OsLCY-b (in the absence of CRTISO)
the two cis isomers were converted within 30 min into 7-cis- and 9-cis-␥-
carotene (peaks 1 and 2, see spectra in supplemental Fig. S4). The elution
profiles in A, B, and C were obtained with HPLC-system 2. lyc., lycopene; car.,
carotene.
Ϫ
sition state. Obviously, a reversible transfer of a single e in a
transient state cannot be excluded either for CRTISO/IDI-2 at
the current state of our knowledge. Although it is possible to
distinguish the different reduced flavin species through UV/Vis
and fluorescence spectroscopy (35, 36) such measurements are
illusory with CRTISO due to sample turbidity caused by lipo-
somes and to the absorption spectra of substrate and products
that lie in the same spectral range (see Fig. 2A).
It is thus not possible as yet to formulate a distinction
between the catalytic mechanisms of CRTY and CRTISO.
Efforts are currently underway to resolve the three-dimen-
sional structure of both enzymes which will hopefully lead to a
clearer picture.
together, the findings presented place CRTISO into a novel
group of flavoproteins catalyzing non-redox reactions with
the help of reduced flavins.
Regional Specificities and Time Course of the cis-to-trans
Isomerization Sequence—Separation and identification of spe-
cific cis-lycopenes formed during catalysis as well as the capa-
bility of converting monocyclic cis-␥-carotene species are in
favor of a “half-side” recognition of the symmetrical tetra-cis-
lycopene substrate. This is mirrored by the predominant for-
mation of the asymmetrically cis-configured intermediate, 7,9-
di-cis-lycopene (peak (b), Fig. 8) that carries cis double bonds in
one-half-side and all-trans double bonds in the other. Onwards
from this intermediate (energetically downhill toward the all-
CRTI, thus indirectly CRTISO, and CrtY are thought to have
evolved during the anoxygenic period of life on Earth (7). It
would therefore appear that such anoxygenic mechanisms of
flavinred catalysis in carotenogenesis are ancient as well. It is,
however, unclear to date how such mechanisms persisted in an
oxygenic atmosphere, i.e. what protective molecular mecha-
nisms co-evolved allowing the use of reduced flavins in aerobic
bacterial cells (CRTY) or in plastids (CRTISO) in close vicinity
of oxygenic photosynthesis.
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CRTISO Catalyzes an FADred-dependent Non-redox Reaction
FIGURE 8. Stereospecific course of reactions catalyzed by CRTISO and specific rates of conversion. The numbers refer to the positions of the cis-double
bonds in the lycopenes shown. (a): 7,9,7Ј,9Ј-tetra-cis-lycopene; (b): 7,9-di-cis-lycopene; (c): 9-cis-lycopene; (d): 7-cis-lycopene; (e): position of cis double bond(s)
unknown; (f): 5-cis-lycopene; (g): all-trans-lycopene. (h): ␤-carotene; LCY-b: lycopene cyclase that introduces ␤-ionone ring groups into the all-trans-lycopene
precursor (g). The k values at the arrows are the rate constants (minϪ1) for a given step as obtained from the simulation procedures. Steps identified by (?)
represent possible, though probably not relevant reaction sequences. The ratios of forward/reverse steps are (a)3(b) ϭ 2; (b) 3 (c) ϭ 0.5; (b) 3 (d) ϭ 0.2;
(b) 3 (e) ϭ 0.15; (b) 3 (f) ϭ 40; (c) 3 (g) ϭ 40; (d) 3 (g) ϭ 60; (e) 3 (g) ϭ 50; (f) 3 (g) ϭ 100.
trans form) the 9-mono-cis (c) and 7-mono-cis (d) intermedi- might be attributed to hitherto unknown secondary parame-
ates can be derived (supplemental Fig. S7). Corroborating this ters, for instance to an altered enzyme-substrate accessibility at
principle of half-side recognition, the symmetric intermediates the enzyme/membrane interface.
7,7Ј-di-cis- and 9,9Ј-di-cis-lycopene or the respective tri-cis-
lycopenes were not found (supplemental Fig. S6).
On the Source of 9-cis Carotenoid Isomers with Altered Bio-
logical Significance—Carotenoids carrying a cis-configured
double bond at position C(9) are frequently found in plant tis-
sues (39). The presence of 9-cis configured xanthophylls has
received special attention since the discovery of the 9-cis epoxy-
carotenoid oxygenases (NCEDs, 1, see Ref. 40 for review) cata-
lyzing the initial step in the biosynthesis of abscisic acid (ABA).
It is therefore tempting to hypothesize that the 9-cis double
bond of violaxanthin and neoxanthin may be a reminiscence of
their (9-cis bearing) prolycopene origin, in which case a regula-
tory role might be attributed to CRTISO. By using isolated 9-cis
and 7-cis-lycopene produced by CRTISO, as well as commer-
cially available 5-cis-lycopene, we show that LCY-b from rice,
encoded by a single gene, acts as a non-permissive selectivity
filter for cis isomers incapable of producing ␤-carotene with
any of these mono-cis-lycopenes (Fig. 7, C and D). Under these
circumstances only monocylic carotenes accumulate. This
finding, if generally applicable, calls for the presence of a yet
unidentified ␤-xanthophyll trans-to-cis isomerase acting at the
C(9) double bond.
The occurrence of 5-cis-lycopene (f) was unexpected because
this cis double bond is derived from a trans-configured one in
the precursor lycopene species indicating the involvement of
a trans-to-cis isomerization step at this position. Quantum
chemistry calculations indicate that the 5-cis isomer of lyco-
pene (f) has the lowest energy among the lycopene cis isomers
considered in this investigation (31). However, its formation
from all-trans-lycopene (step g Ͼ f, k18 in Fig. 8) is assumed to
occur at a very low rate due to the predicted presence of a large
rotational energy barrier (31). In the presence of CRTISO, this
isomer (f) was found to be formed from poly-cis configured
precursors but not from all-trans-lycopene (supplemental Fig.
S8). The biological role of this reaction remains enigmatic.
There may be significance with respect to the lycopene ⑀-cy-
clase reaction based on a consideration by Eugster (37, 38)
according to which the cis configuration of the C(5)-C(6) dou-
ble bond of lycopene together with the mode of folding of the
ring precursor (chair or boat) determine the feasibility of axial
proton abstraction and thus the formation of the ⑀-ionone ring.
CRTISO establishes an equilibrium between cis isomers
(Figs. 5 and 8). In accordance with this, the addition of LCY-b
disrupts the equilibrium by catalyzing the essentially irreversi-
ble cyclization of all-trans-lycopene (g) to ␤-carotene (h). Con-
sequently, all cis isomers (a)-(f) are rapidly converted into all-
trans lycopene (Fig. 6). However, upon incubation of CRTISO
with all-trans-lycopene (g), the expected reversibility of the
reaction could not be detected (supplemental Fig. S8). This
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