From the Department of Plant Molecular Physiology, University of Hawaii-Manoa, Honolulu, Hawaii 96822
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Violaxanthin de-epoxidase and zeaxanthin
epoxidase catalyze the addition and removal of epoxide groups in
carotenoids of the xanthophyll cycle in plants. The xanthophyll cycle
is implicated in protecting the photosynthetic apparatus from excessive
light. Two new sequences for violaxanthin de-epoxidase from tobacco and Arabidopsis are described. Although the mature proteins are
well conserved, the transit peptides of these proteins are divergent, in contrast to transit peptides from other proteins targeted to the
thylakoid lumen. Sequence analyses of both violaxanthin de-epoxidase and zeaxanthin epoxidase establish the xanthophyll cycle enzymes as
members of the lipocalin family of proteins. The lipocalin family is a
diverse group of proteins that bind small hydrophobic (lipophilic)
molecules and share a conserved tertiary structure of eight -strands
forming a barrel configuration. This is the first reported
identification of lipocalin proteins in plants.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The xanthophyll cycle is comprised of de-epoxidation and epoxidation interconversions of three xanthophylls (violaxanthin, antheraxanthin, and zeaxanthin), catalyzed by two enzymes that are localized on opposite sides of the thylakoid membrane. Violaxanthin de-epoxidase (VDE)1 is localized in the lumen of thylakoids and catalyzes de-epoxidation of violaxanthin in the presence of ascorbate and an acidic lumen, the latter formed by the light-induced proton pump (1-5). Zeaxanthin epoxidase is localized on the stromal side of the thylakoid membrane and catalyzes the epoxidation of zeaxanthin in the dark or under low light intensities (6, 7). The reaction is optimal near pH 7.5, and the enzyme utilizes oxygen, ferredoxin, and FAD as co-substrates (6-10). A role for zeaxanthin was first described in 1987 by Demmig et al. (11) to protect the photosynthetic apparatus against the adverse effects of excessive light. Recent evidence demonstrates that accumulation of both antheraxanthin and zeaxanthin, along with the transthylakoid pH gradient, mediates the non-radiative dissipation of excess energy as heat (12-17). The xanthophyll cycle is thought to have evolved early in the development of higher plants as it is present in all plants examined thus far (18).
Pervaiz and Brew (19, 20) first identified a group of proteins, based
on sequence homology, that have a common role in binding and transport
of small hydrophobic molecules. These proteins, designated the
lipocalins, represent a diverse group of proteins from the animal
kingdom (for review see Ref. 21) and recently from a prokaryote (22).
These lipocalin proteins have a common tertiary structure of an
eight-stranded anti-parallel -barrel, and only one protein to date
displays catalytic activity. We report that violaxanthin de-epoxidase
and zeaxanthin epoxidase are members of the lipocalin family. To our
knowledge, they are the first lipocalins described from plants and only
the second reported to demonstrate enzymatic activity. The deduced
polypeptide sequences of three VDE proteins are compared, and the
transit peptides are analyzed against other thylakoid lumen
proteins.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA Library Construction and Screening--
A cDNA
library was constructed from poly(A)+ RNA isolated from a
pooled sample of young tobacco (Nicotiana tabacum cv.
Xanthi) leaves using the Timesaver cDNA synthesis kit (Amersham
Pharmacia Biotech) and ligated into ZAPII (Stratagene). The
Arabidopsis cDNA library (designated as
PRL2) was
obtained from the Arabidopsis Biological Resource Center at Ohio State
University. The library was derived from pooled mRNA of various
tissues of the Columbia wild type of Arabidopsis thaliana
(L.) Heynh. The cDNA libraries were screened using a random primed
32P-labeled probe prepared from the coding region of the
lettuce violaxanthin de-epoxidase cDNA (23). Hybridization was
performed according to Church and Gilbert (24) using a hybridization
and washing temperature of 55 °C. Strongly hybridizing cDNAs
were excised according to the manufacturer's instructions.
Sequencing-- Both strands of cDNA were sequenced completely using an Applied Biosystems model 373A automated sequencer.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Violaxanthin de-epoxidase cDNAs were isolated and sequenced from Arabidopsis and tobacco. Analysis of the deduced polypeptide sequences of mature VDE proteins indicates high conservation among dicotyledonous plants (Fig. 1). Tobacco VDE shares 91.6 and 89.8% similarity and 82.6 and 82.0% identity with the Arabidopsis and lettuce VDE proteins, respectively. In the mature proteins only nine amino acid positions have different amino acids in all three proteins. All three proteins have a cysteine-rich domain, a lipocalin signature, and a highly charged domain as described previously for the lettuce VDE (23). The 13 cysteine residues that were reported for the lettuce sequence are invariant in all three proteins. Cysteines are functionally important because dithiothreitol is an inhibitor of VDE (25). Partial inhibition of VDE activity with low dithiothreitol concentrations results in an accumulation of antheraxanthin (13) suggesting that there is more than one disulfide linkage related to activity. One difference between the amino acid sequences is that the lettuce and Arabidopsis VDE have a highly charged peptide repeat (Glu-Val-Glu-Lys) whereas the tobacco sequence does not (underlined sequences in Fig. 1). The significance of this duplication is unknown because it does not appear to affect enzyme activity.
|
Earlier analysis demonstrated that the lettuce VDE transit peptide is a typical bipartite type with an N-terminal portion representing a transit peptide for targeting to the chloroplast stroma followed by a short hydrophobic thylakoid targeting signal peptide-like domain at the C-terminal end of the peptide (23, 26). Also, the cleavage site for the lettuce VDE was determined from the N-terminal sequence of purified VDE from lettuce (27). A comparison of the transit peptides for the three VDE proteins reveals no strong overall homology in primary structure except for the N- and C-terminal ends (Fig. 1). The transit peptides have a mean (±S.D.) percent similarity and identity of 49.3 ± 4.1 and 24.8 ± 2.2, respectively. We performed sequence comparisons on transit peptides from six plastocyanin, six 23-kDa polypeptides, and five 33-kDa polypeptides of the oxygen-evolving complex associated with photosystem II; all are proteins targeted to the thylakoid lumen. Mean percent similarities of 73.4 ± 7.3, 70.4 ± 8.7, and 80.4 ± 4.4 and mean percent identities of 60.6 ± 7.7, 60.4 ± 8.7, and 63.4 ± 5.8 were calculated for plastocyanin and 23- and 33-kDa polypeptides of the oxygen-evolving complex, respectively. In addition, the transit peptides for three zeaxanthin epoxidases, a protein targeted to the chloroplast stroma, have a mean percent similarity and identity of 77.5 ± 12.4 and 70.1 ± 15.4, respectively. The values for similarity and identity among the VDE transit peptides are significantly lower than for the other transit peptides discussed, indicating that the VDE transit peptides are more divergent between plant species. Despite this divergence in the primary structure, the hydropathy profiles for the transit peptides are quite similar (Fig. 2). The transit peptides are mostly hydrophilic except for the C-terminal end, which has a characteristic short hydrophobic region. Another difference is the variable lengths of VDE transit peptides. It was reported earlier that similar proteins in different organisms have comparable transit peptide lengths (29). For example, six plastocyanin sequences, five 33-kDa polypeptides, and six 23-kDa polypeptides of the oxygen-evolving complex associated with photosystem II, all lumenal proteins, have transit peptide lengths of 66-72, 79-85, and 73-82 residues, respectively. However, the three transit peptides for VDE have a range of 113-134 amino acids. Not only are the VDE transit peptides more variable in length, but they are overall significantly longer than the transit peptides from the other lumenal proteins compared above.
|
Two very short regions of local homology are observed in the VDE
transit peptides at both ends of the peptide (Fig. 1). At the N
terminus all three VDE transit peptides start with a Met-Ala dipeptide.
Earlier analysis of 26 transit peptides of stromal proteins revealed
that 85% had this same N-terminal dipeptide (30). Moreover, 18 thylakoid lumen proteins from various dicot plants including
plastocyanin and the 16-, 23-, and 33-kDa polypeptides of the
oxygen-evolving complex associated with photosystem II show this same
N-terminal Met-Ala dipeptide. This dipeptide probably signals the
removal of the N-terminal methionine (30-32). Most eukaryotic proteins
lose their N-terminal methionine co- or post-translationally by a
methionine aminopeptidase, especially when the methionine is followed
by an alanine. The resulting N-terminal alanine can then be modified by
acetylation (31, 32). At the cleavage site for the three VDE transit
peptides, all exhibit a short homologous region having a consensus
sequence of (Ala/Val)-Asp-AlaVal-Asp. This fits the consensus
pattern for the cleavage site in the precursors of proteins of the
thylakoid lumen (Ala-X-Ala
) and is the same type of
cleavage site identified in bacterial signal peptides (30, 33). In
addition, the thylakoid lumen protein plastocyanin not only has a very
similar consensus (Ala-(Met/Leu)-Ala
hydrophobic-(Asp/Glu)) as VDE
but also has a similar N terminus of the mature protein starting with a
hydrophobic residue followed by an acidic residue.
While performing a data base search, we identified an almost identical alignment of the Arabidopsis VDE cDNA with a genomic clone containing the Arabidopsis syntaxin-related KNOLLE gene (GenBank accession no. U39452). This gene maps to chromosome 1 in Arabidopsis and is located next to the g5957 restriction fragment length polymorphism marker (34). The Arabidopsis VDE gene is located in the 3'-untranslated region of the KNOLLE gene and is in the opposite orientation. According to the polyadenylation sites of both genes, there is at most 141 nucleotides separating the two genes. In addition, we identified an identical match to Arabidopsis VDE in a large genomic fragment from Arabidopsis chromosome 1 recently released to the data base (GenBank accession no. AC003981). It contains the entire VDE sequence along with the promoter. According to the sequence alignments between the cDNA and genomic DNA, the VDE gene contains four introns ranging in size from 77 to 265 nucleotides.
Prior analysis of the deduced polypeptide sequence of lettuce VDE
identified a lipocalin signature motif (23), and the polypeptide sequences from tobacco and Arabidopsis have this motif as
well. The lipocalins are a group of diverse proteins characterized by their ability to bind small hydrophobic (lipophilic) molecules (19,
20). Although they were originally thought to function only as
transport proteins, members of this group have a wide range of roles
including retinol transport, cryptic coloration, olfaction, pheromone
transport, enzymatic synthesis of prostaglandins, immunomodulation, and
regulation of cell homeostasis (21). Although lipocalins have great
sequence dissimilarity with sequence identity often falling below 20%
(35), x-ray crystallographic structures of lipocalins exhibit a highly
conserved folding pattern (36-39). Based on these structures, the
lipocalin fold is characterized as a single eight-stranded
anti-parallel -sheet (Fig. 3) with the
overall shape resembling a flattened cone-shaped barrel or calyx (37,
39). The
-barrel encloses an internal ligand binding site and also
encloses the bound hydrophobic molecule and minimizes solvent contact
(20, 35).
|
Because the xanthophyll cycle enzymes have a common substrate (antheraxanthin), it is reasonable to consider that zeaxanthin epoxidase may have a similar tertiary structure to VDE. The zeaxanthin epoxidase gene was recently identified from an abscisic acid-deficient mutant of Nicotiana plumbagnifolia that was impaired in zeaxanthin epoxidation (40). The resulting zeaxanthin epoxidase cDNA was also used to isolate cDNAs from pepper (10) and tomato (41). A motif search using the GCG sequence analysis software was performed with the three deduced polypeptide sequences for zeaxanthin epoxidase, but no lipocalin signature motif was detected. However, after closer examination of the sequences, a lipocalin signature motif can be identified in all three sequences. The lipocalin motif in the zeaxanthin epoxidase sequences has some slight differences that are not accounted for by the consensus sequence for the lipocalin signature motif.
The lipocalins are classified as kernel or outlier based on homology of
three motifs from three structurally conserved regions of the lipocalin
fold (21, 35, 42). The three structurally conserved regions are
localized in: (a) the first -strand and a short
310-helix preceding this
-strand; (b)
portions of the sixth and seventh
-strands including the connecting
loop; and (c) a portion of the eighth
-strand including
the loop and part of the C-terminal
-helical structure (Fig. 3). In
the folded protein these three regions are in close proximity to one
another and are localized on one side of the barrel (21, 42). The first
and largest group is known as the kernel lipocalins and shares homology
in all three motifs whereas the other group is known as the outlier
lipocalins and is more divergent because significant homology is only
observed in motif I (the lipocalin signature). Outlier lipocalins
exhibit weaker homology in motifs II and III in contrast to the kernel
lipocalins. Sequence alignments of the motifs in a number of lipocalins
from diverse species including mammalian, crustacean, insect, and
prokaryote illustrate this homology (Fig. 3). Motif I is well conserved
in all sequences with the key features of an invariant Gly followed by
a positively charged residue in most cases and an invariant Trp
followed by a residue with a ring structure. Included in these
comparisons are two outlier proteins (human
1-acid
glycoprotein and rat von Ebner's gland protein) exhibiting homology in
motif I but somewhat less homology in motifs II and III. Also included
in this motif comparison is the enzyme prostaglandin D synthase, the
only known lipocalin to date to have catalytic activity. Here we
introduce violaxanthin de-epoxidase and zeaxanthin epoxidase to the
lipocalin family. They are the first lipocalins identified from plants
and are unique in that they also have catalytic activity. These enzymes of the xanthophyll cycle share homology in the structurally conserved regions especially in motif I with the invariant Gly and Trp present. Homology in motifs II and III is much weaker than in kernel lipocalins, suggesting that these enzymes would fall in the class of outlier lipocalins.
To further analyze whether the xanthophyll enzymes fit the overall
structure of the lipocalin model, the distances between the three
motifs were compared for the various lipocalin proteins. Both kernel
and outlier lipocalins have the same characteristics with respect to
the spacing of the motifs. Typically, the distances between motifs I
and II range from 65 to 73 amino acids whereas the range between motifs
II and III is 10-17 amino acids (Fig. 4). The tobacco VDE fits the pattern of
other lipocalins with distances of 72 and 15 amino acids between motifs
I and II and motifs II and III, respectively. The main difference
between VDE and the other lipocalins is the long C-terminal tail
following motif III. Secondary structure predictions using the PHDsec
program (43-45) detected six -strands in the tobacco VDE within the
region spanning the lipocalin motifs. Secondary structure predictions using this program also detected six
-strands in retinol-binding protein, a lipocalin protein known to have eight
-strands within the
region spanning the lipocalin motifs as determined by x-ray crystallography (36). The cysteine-rich N-terminal portion of VDE is
predicted to consist of
-helices, and the C-terminal tail with its
high concentration of glutamic acid residues is predicted to be a
series of long
-helices. Partial protonation of these glutamic acid
residues near pH 5.0, the optimal pH for VDE activity (2, 46), may play
a role in binding to the thylakoid membrane (23). Zeaxanthin epoxidase,
however, is different in that the distance between motifs I and II is
103 amino acids, much greater than the range for the lipocalin proteins
(Fig. 4). This suggests that zeaxanthin epoxidase could have more than
eight
-strands or possibly longer loop structures. The distance
between motifs II and III falls into the range of the other lipocalins.
The PHDsec program for secondary structure (43-45) predicts eight
-strands within the region spanning the lipocalin motifs for tobacco
zeaxanthin epoxidase, two more than was detected for tobacco VDE and
retinol-binding protein. The remaining N- and C-terminal regions of the
tobacco zeaxanthin epoxidase are predicted to consist of both
-helices and
-strands with a predominance of
-helices.
Although zeaxanthin epoxidase fits the description of a lipocalin
protein based on motif homology, the distance between motifs I and II
suggests the protein may have a somewhat modified structure.
|
Nearly two decades ago, a narrow well-like cavity structure was
proposed for the active site of VDE based on activity against various
substrates (5). Substrates with all-trans configurations in
the acyclic polyene chain (violaxanthin, antheraxanthin, neoxanthin, and diadinoxanthin) were all de-epoxidized by VDE whereas no
de-epoxidation was observed with substrates having a 9-cis
configuration. This suggests the putative -barrel structure is
narrow, allowing only the entry of all-trans pigments. Based
on the length of the xanthophyll substrates in the 9-cis
configuration, the length of the cavity was approximated minimally at
30 Å deep (5). This is within reason because the x-ray
crystallographic structures of the lipocalin proteins insecticyanin and
retinol-binding protein indicate that both are approximately 40 Å deep
(36, 38). The evolutionary implications of the
-barrel structure
suggest that it may be well suited for small hydrophobic molecules
because the barrel internally provides a hydrophobic environment for
the ligand. For example, analysis of x-ray crystal structures shows
that the binding pocket of insecticyanin is decidedly hydrophobic (38), and the binding pocket for retinol-binding protein is composed of both
hydrophobic and uncharged residues (36). Analysis of the xanthophyll
enzymes to determine the amino acid residues important in binding and
catalytic activity to help further our understanding of the role of
these enzymes in photoprotection in plants is now warranted.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Department of Energy Division of Biosciences Grant DE-FG03-92ER20078 and United States Department of Agriculture National Research Initiative Competitive Grant 97-35100-4851.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U34817 and U44133.
To whom correspondence should be addressed: Dept. of Plant
Molecular Physiology, University of Hawaii, 3190 Maile Way, Honolulu, HI 96822. Tel: 808-956-7934; Fax: 808-956-3542; E-mail: yamamoto{at}hawaii.edu.
1 The abbreviation used is: VDE, violaxanthin de-epoxidase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|