Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups

Biochemical Society Transactions (2000) Volume 28, part 6
thus indicating that although pericarps constitute
References
the bulk of the mass of coarse corn ?bre, they
1 Moreau, R. A., Powell, M. J. and Hicks, K. B. (1996) J. Agric.
contribute little to the composition of corn-?bre
Food Chem. 44, 2149–2154
oil.
2 Moreau, R. A., Singh, V., Eckhoff, S. R., Powell, M. J., Hicks,
K. B. and Norton, R. A. (1999) Cereal Chem. 76, 449–451
3 Seitz, L. M. (1989) J. Agric Food. Chem. 37, 662–667
Mention of a brand or firm name does not constitute an
endorsement by the U.S. Department of Agriculture above others
of a similar nature not mentioned.
Received 23 June 2000
Molecular evolution of lycopene cyclases involved in the formation of carotenoids
with ionone end groups
P. Krubasik and G. Sandmann1
Biosynthesis Group, Botanical Institute, J. W. Goethe Universita$t, P.O. Box 111930, D-60054 Frankfurt, Germany
Abstract
?-ionone rings. The enzymes involved in these
A survey is given of the lycopene cyclase genes
cyclization reactions are lycopene cyclases. The
present in bacteria, fungi and plants where two
genes of lycopene cyclases have been cloned from
completely unrelated types exist. One is the
many bacteria, from fungi and from plants.
classical monomeric bacterial ?-cyclase gene, crtY,
which may be an ancestor of crtL, the gene for a
The classical monomeric lycopene
?-cyclase in cyanobacteria. From crtL a line of
cyclases of plants and many bacteria
evolution can be drawn to plant ?- and ?-cyclase
The ?rst lycopene cyclase gene, crtY, was cloned
genes and to the gene of capsanthin\capsorubin
from the eubacterium Erwinia uredovora [1]. Sev-
synthase. In Gram-positive bacteria two genes
eral other very similar crtY genes are now available
crtYc and crtYd are present. They encode two
from other non-photosynthetic bacteria. So far the
proteins which have to interact as a heterodimer
only cyanobacterial lycopene cyclase gene (crtL) is
for lycopene ?-cyclization. From this type of
known from Synechococcus sp. PCC7942 [2]. Its
lycopene cyclase gene the fungal lycopene cyclase\
similarity to the bacterial crtY-type cyclases is
phytoene synthase fusion gene evolved.
rather low. Nevertheless, distinct conserved pat-
terns in the amino acid sequence can be observed
Introduction
in crtY, crtL and the lycopene cyclase genes from
Carotenoids are widely distributed in Nature and
plants (Figure 1). These are one hypothetical
more than 600 di?erent structures are known.
dinucleotide-binding region with similarities to
Plants, several bacteria and some fungi are able to
the one found in phytoene desaturases [3] (Figure
synthesize carotenoids. Even though the end pro-
1, box A) and four further motifs with unknown
ducts of carotenoid biosynthesis can be very
function [4] (Figure 1, boxes B–E). A common
diverse, a general common pathway leading to the
phylogenetic origin of crtY, crtL and lycopene
formation of cyclic ?-carotene can be observed in
cyclase genes from plants is therefore assumed [5].
many prokaryotic and eukaryotic organisms. The
The amino acid sequence of lycopene cyclases
synthesis of this C
from plants and cyanobacteria are closely related,
%! carotene starts by the con-
densation of two molecules of geranylgeranyl
as indicated by the phylogenetic tree (Figure 2A).
pyrophosphate followed by four desaturation
All these lycopene cyclases are polypeptides with
steps. Then, the ends of the resulting acyclic
around 400 amino acids and a molecular mass of
lycopene may be cyclized to ?-ionone, ?-ionone or
43 kDa [6], and the enzymes from plants have an
additional N-terminal transit sequence of 100
amino acids.
Key words : capsanthin/capsorubin synthase, fungal lycopene
The formation of ?-ionone and ?-ionone rings
?-cyclase, heterodimeric lycopene cyclase, lycopene-? cyclase,
in plants is catalysed by two di?erent enzymes,
lycopene cyclase/phytoene synthase fusion.
?-cyclase and ?-cyclase. Both enzymes show high
Abbreviation used : Ccs, capsanthin capsorubin synthase.
1To whom correspondence should be addressed (e-mail
similarities in their amino acid sequence and it is
Sandmann!em.uni-frankfurt.de).
very likely that they evolved from the same
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Sterols and Isoprenoids
Figure 1
Comparison of the amino acid sequences of lycopene ?-cyclases from bacteria (CrtY), cyanobacteria (CrtL) and
plants (Lcyb), and of ?-cyclases from plants (Lcye)
In the upper panel the locations of five conserved regions are shown ; the lower panel shows sequence alignments of seven different
lycopene cyclases in these regions. A.t, Arabidopsis thaliana ; L.e, Lycopersicon esculentum ; S.7942, Synechococcus sp. PCC7942 ; S.g,
Streptomyces griseus ; E.u, Erwinia uredovora ; AA, amino acids.
ancestor. Another member of this protein family
The new heterodimeric lycopene
of plant lycopene cyclases is the capsanthin capso-
cyclases
rubin synthase (Ccs) from Capsicum annuum [7].
A second type of lycopene cyclase was recently
This enzyme converts antheraxanthin or vio-
described for the Gram-positive coryneform bac-
laxanthin into capsanthin or capsorubin by a
terium Brevibacterium linens [9]. In this new type
mechanism similar to lycopene cyclization [8].
of lycopene cyclase the two di?erent genes crtYc
In addition, Ccs can also convert lycopene into
and crtYd code for two small polypeptides of
?-carotene and the amino acid sequences of Ccs
125 and 107 amino acids, respectively. Only the
and lycopene cyclases are very similar. Therefore
combined products of both genes are able to con-
it was proposed that ccs evolved from a duplicated
vert lycopene into ?-carotene. Thus the functional
lycopene cyclase gene.
807
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Biochemical Society Transactions (2000) Volume 28, part 6
Figure 2
(A) Unrooted phylogenetic tree of monomeric lycopene cyclases and (B) amino acid sequence alignments of
heterodimeric lycopene cyclases
(A) Ccs from Citrus sinensis and CrtY from Deinococcus radiodurans were determined by sequence similarities ; their functions in carotenoid
biosynthesis have not been shown. (B) Amino acid sequence alignments of heterodimeric lycopene cyclases from Mycobacterium marinum
(M.m), Mycobacterium aurum (M.a), Brevibacterium linens (B.l), Myxococcus xanthus (M.x) and the N-terminal parts of the bifunctional
lycopene cyclases/phytoene synthases from Neurospora crassa (N.c), Mucor circinelloides (M.c) and Xanthophyllomyces dendrorhous (X.d).
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Sterols and Isoprenoids
enzyme is referred to as heterodimeric lycopene
organisms and has N-terminal similarities to the
cyclase. The genes of these lycopene cyclases show
new heterodimeric lycopene cyclases CrtYc and
no sequence similarities with any known genes or
CrtYd from B. linens (Figure 2B). Owing to the
with any of the conserved regions of the classical
bifunctionality of its gene product, the gene was
lycopene cyclases. The same type of cyclases has
called crtYB. By truncation of the crtYB cDNA it
been functionally identi?ed in the carotenoid gene
could be demonstrated that its phytoene synthase
cluster of Mycobacterium aurum [10] and similar
and lycopene cyclase domains are localized in the
sequences were found near a functionally identi-
regions of the polypeptide with sequence similar-
?ed phytoene synthase gene in the genome of
ities to phytoene synthases and the new lycopene
Mycobacterium marinum [11]. Genome sequencing
cyclases, respectively. The gene product of al-2
data also reveals a similar lycopene cyclase for
from Neurospora crassa, which has been shown to
Mycobacterium avium (http :\\www.tigr.org, data-
be involved in phytoene synthesis of this asco-
base m-avium, contig 89). As shown in the
mycetous fungus [15], has a high overall sequence
alignments of Figure 2(B), these genes are highly
similarity to CrtYB from X. dendrorhous. Like
homologous and share several conserved regions.
CrtYB the al-2 gene product is not only a phytoene
None of them could be found in the crtY genes.
synthase but also exhibits lycopene cyclase activity
All species possessing the crtYc and crtYd
(P. Krubasik and G. Sandmann, unpublished
genes belong to the order Actinomycetales, a group
work). By database searches a further sequence
of Gram-positive bacteria with a high GC content
from the zygomycetous fungus Mucor circi-
in their DNA. Surprisingly, in Streptomyces gris-
nelloides with similarities to CrtYB was found
eus, also belonging to the Actinomycetales, a lyco-
and it can be assumed that it also encodes a bi-
pene cyclase of the classical CrtY-type has been
functional lycopene cyclase\phytoene synthase.
functionally
identi?ed
[12].
The
sequencing
Moreover, based on classical genetic studies, a
project
of
Streptomyces
coelicolor
(http :\\
common translation or a fusion of lycopene
www.sanger.ac.uk.\projects\S-coelicolor, cosmid
cyclase and phytoene synthase has been predicted
StJ12) also revealed the presence of a classical
for M. circinelloides [16].
lycopene cyclase. In summary, the carotenoid-
Lycopene cyclase genes from all big groups of
producing Actinomycetales seem to have the new
phylogenetically related fungi, basidiomycetes,
type of heterodimeric lycopene cyclase, with the
ascomycetes, and zygomycetes, were found. As
exception of Streptomyces species, which have
the ?rst two groups developed from forms of the
the classical CrtY-type lycopene cyclase. Interest-
latter, it can be supposed that this unique type of
ingly, the carotenoid gene cluster of the Gram-
lycopene cyclase\phytoene synthase must have
negative bacterium Myxococcus xanthus also has
been acquired relatively early in the evolution of
genes with similarities to crtYc and crtYd from
fungi. The bifunctional lycopene cyclase might
B. linens [13]. Even though a function could
have originated from the fusion of two crtYc- and
not be assigned to these two genes, it can be
crtYd-type genes, similar to those in B. linens,
expected that they encode the lycopene cyclase
and from a phytoene synthase. The fusion of crtYc,
of
this
organism,
which
synthesizes
cyclic
crtYd and the phytoene synthase gene might have
carotenoids.
occurred by recombinant processes. An example
of how a carotenogenic fusion gene could originate
by chromosomal rearrangement was recently
The bifunctional fungal lycopene
given [17]. In a Rubrivivax gelatinosous strain with
cyclases
an inactivated crtB gene, which therefore is devoid
The ?rst lycopene cyclase gene from a fungus was
of carotenoids, illegitimate recombination of a new
isolated and identi?ed from the heterobasidio-
functional chimaeric crtB gene was forced by
mycetous yeast Xanthophyllomyces dendrorhous
photo-oxidative stress.
[14]. Its cDNA encodes a bifunctional lycopene
cyclase\phytoene synthase of 673 amino acids.
Other types of lycopene cyclase
This gene product has two catalytic activities in
At least one further type of lycopene cyclase has to
carotenoid biosynthesis as it converts geranyl-
be expected, as sequence comparisons of the
geranyl phosphate into phytoene as well as lyco-
known lycopene cyclases with the genome of
pene into ?-carotene. The deduced amino acid
the fully sequenced cyanobacterium Synechocystis
sequence of this cDNA has similarities in its
sp. PCC6804 [18] did not result in a signi?cant
C-terminal part to phytoene synthases from other
match with any known lycopene cyclase. A lyco-
809
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Biochemical Society Transactions (2000) Volume 28, part 6
pene cyclase gene must exist in this organism be-
3 Armstrong, G. A., Hundle, B. S. and Hearst, J. E. (1993)
cause ?-carotene, which is the product of lycopene
Methods Enzymol. 214, 297–311
4 Cunningham, Jr, F. X., Pogson, B., Sun, Z., McDonald, K. A.,
cyclization, was found in this cyanobacterium
DellaPenna, D. and Gantt, E. (1996) Plant Cell 8,
[19]. The genomic sequence data of one further
1613–1626
completely sequenced cyanobacterium, Anabaena
5 Hugueney, P., Badillo, A., Cehn, H.-C., Klein, A., Hirschberg,
sp. PCC7120 (http :\\www.kazusa.or.jp\cyano\
J., Camara, B. and Kuntz, M. (1995) Plant J. 8, 417–424
anabaena), did also lack similarities to the
6 Schnurr, G., Misawa, N. and Sandmann, G. (1996) Biochem.
sequences of known lycopene cyclase genes.
J. 315, 869–874
7 Bouvier, F., Hugueney, P., d ’Harlingue, A., Kuntz, M. and
Camara, B. (1994) Plant J. 6, 45–54
Conclusion on the phylogeny of
8 Bouvier, F., d’Harlingue, A. and Camara, B. (1997) Arch.
lycopene cyclase genes
Biochem. Biophys. 346, 53–64
Two completely unrelated lycopene ?-cyclases
9 Krubasik, P. and Sandmann, G. (2000) Mol. Gen. Genet.
evolved independently in bacteria. The hetero-
263, 423–432
10 Viveiros, M., Krubasik, P., Houssaini-Iraqui, M., Sandmann, G.
dimeric lycopene ?-cyclase encoded by the genes
and David, H. L. (2000) FEMS Microbiol. Lett., 187, 95–101
crtYc and crtYd dominates among Gram-positive
11 Ramakrishnan, L., Tran, H. T., Federspiel, N. and Falkow, S.
bacteria. From these genes the fungal lycopene
(1997) J. Bacteriol. 179, 5862–5868
?-cyclase\phytoene synthase fusion gene crtYB
12 Kru$gel, H., Krubasik, P., Weber, K., Saluz, H. P. and
derived. The completely unrelated crtY of a mono-
Sandmann, G. (1999) Biochim. Biophys. Acta 1439, 57–64
meric ?-cyclase, identi?ed ?rst in a Gram-negative
13 Botella, J. A., Murillo, F. J. and Ruiz-Vazquez, R. (1995) Eur. J.
Biochem. 233, 238–248
enterobacterium, may be an ancestor of a cyano-
14 Verdoes, J. C., Krubasik, P., Sandmann, G. and van Ooyen,
bacterial and prochlorophyte lycopene ?-cyclase
A. J. J. (1999) Mol. Gen. Genet. 262, 453–461
gene crtL. From this gene, a line of evolution can
15 Schmidhauser, T., Lauter, F., Schumacher, M., Zhou, W.,
be drawn to the corresponding lcy-? genes in
Russo, V. E. A. and Yanofsky, C. (1994) J. Biol. Chem. 269,
chlorophytes and higher plants. The genes en-
12060–12066
16 Velayos, A., Lo!pez-Matas, M. A., Ruiz-Hidalgo, M. J. and
coding lycopene ?-cyclase, lcy-?, and Ccs, ccs, from
Eslava, A. P. (1997) Fungal Genet. Biol. 22, 19–27
plants are highly homologous with the ?-cyclase
17 Ouchane, S., Picaud, M., Vernotte, C. and Astier, C. (1997)
genes and may originate from gene duplications.
EMBO J. 16, 4777–4787
18 Kotani, H., Tanaka, A., Kaneko, T., Sato, S., Sugiura, M. and
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Received 22 June 2000
Effect of monogalactosyldiacylglycerol and other thylakoid lipids on violaxanthin
de-epoxidation in liposomes
D. Latowski, A. Kostecka and K. Strza?ka1
Department of Plant Physiology and Biochemistry, The Jan Zurzycki Institute of Molecular Biology,
Jagiellonian University, Al. Mickiewicza 3, 31-120 Krako!w, Poland
Abstract
phosphatidylcholine vesicles supplemented with
In this study we present evidence that one of two
monogalactosyldiacylglycerol (MGDG). Activity
reactions of the xanthophyll cycle, violaxanthin
of violaxanthin de-epoxidase (VDE) in this system
de-epoxidation, may occur in unilamellar egg
was found to be strongly dependent on the content
of MGDG in the membrane ; however, only to a
level of 30 mol %. Above this concentration the
Key words : violaxanthin de-epoxidase, xanthophyll cycle.
rate of violaxanthin de-epoxidation decreased.
Abbreviations used : VDE, violaxanthin de-epoxidase ; PC,
The e?ect of individual thylakoid lipids on VDE-
phosphatidylcholine ;
MGDG,
monogalactosyldiacylglycerol ;
independent violaxanthin transformation was also
DGDG, digalactosyldiacylglycerol ; SQDG, sulphoquinovosyldi-
investigated and unspeci?c e?ects of phos-
acyglycerol ; PG, phosphatidylglycerol.
1To whom correspondence should be addressed (e-mail
phatidylglycerol and sulphoquinovosyldiacygly-
strzalka!mol.uj.edu.pl).
cerol, probably related to the acidic character of
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