(Received for publication, March 13, 1995; and in revised form, May 16, 1995)
From the
Carotenoids have recently received considerable interest because
of their potential in delaying or preventing degenerative diseases such
as arteriosclerosis, cancer, and aging. In this study we show that the
active oxygen species singlet oxygen (
The carotenoid astaxanthin
(3,3`-dihydroxy- The yeast P.
rhodozyma could serve as an excellent microbial model to study the
functions of astaxanthin and other carotenoids in cell aging.
Astaxanthin is an extremely potent antioxidant, and its antioxidant
activity has been reported to be stronger and to last longer than
carotenoids naturally present in vegetables including
Yeasts
were grown as described previously(6, 11) . Briefly, 5
ml of YM broth was inoculated with cells from slants and the yeasts
were grown aerobically in roller drum tubes at 20 °C for 2 days
until a dry cell weight of
In certain experiments, cultures were fed with
[2- Cultures were
also exposed to chemically generated
Figure 1:
Carotenoid content in P.
rhodozyma strain 67-385 exposed to photochemically generated
We were interested if brief
exposure of the inoculum to
Figure 2:
Oxygen uptake by P. rhodozyma strain 67-385 and sensitivity to 1.3 mM KCN as a function
of culture age.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
O
) and
peroxyl radicals differently affect carotenoid composition and
biosynthesis in the yeast Phaffia rhodozyma. Photochemical
generation of
O
with rose bengal or
-terthienyl induced carotenoid accumulation. In contrast, peroxyl
radicals derived from t-butylhydroperoxide (tBOOH) or
H
O
decreased the content of astaxanthin and
increased
-carotene by
4-fold, suggesting end product
feedback regulation by astaxanthin or inhibition of biosynthetic
enzymes.
C labeling of carotenoids during oxidative stress
supported the possibility of end product regulation. Carotenoids were
bleached by 8 mM tBOOH within 6 h when carotenogenesis was
inhibited by thymol. When treated with peroxides, a previously
unreported pigment in P. rhodozyma was formed. The carotenoid
had a mass of 580 Da and a molecular formula of
C
H
O
. Chemical derivatizations
combined with mass and absorbance spectroscopy tentatively identified
the carotenoid as dehydroflexixanthin
(3,1`-dihydroxy-2,3,3`,4`-tetradehydro-1`,2`-dihydro-
,
-carotene-4-one).
This study provides the first report of induction of astaxanthin
biosynthesis by
O
, probable feedback control by
astaxanthin, and the oxidative degradation of astaxanthin to novel
pigments in P. rhodozyma.
,
-carotene-4,4`-dione) has attracted
considerable interest in recent years because of its potent antioxidant
activity and possible role in delaying or preventing degenerative
diseases(1, 2) , and also because of the economic
value of astaxanthin as a pigment source in salmonid
aquaculture(3) . Only a few species of microorganisms produce
astaxanthin in nature. Of these limited biological sources, the
heterobasidiomycetous yeast Phaffia rhodozyma(4) has
attracted the interest of several fermentation companies because it
produces astaxanthin as its major carotenoid and attains high levels of
biomass (>50 g of dry yeast/liter) in fermentor culture(5) .
The fermentation industry has focused on strain development and
fermentor control for maximum production of astaxanthin, and limited
research has been devoted to the genetics and enzymology of astaxanthin
biosynthesis/degradation and of its physiological roles in the yeast.
We previously showed that carotenoids protect P. rhodozyma against killing by singlet oxygen (
O
)(
)(6) , and that
carotenoids provide a selective advantage to pigmented strains compared
to albino mutants. This finding appeared reasonable because the natural
habitat of P. rhodozyma is sap fluxes of the birch tree Betula, and the host tree contains an unidentified compound
that catalyzes the formation of
O
on exposure
to ultraviolet light(6) . Carotenogenesis may have evolved in P. rhodozyma to ensure survival in face of photogenerated
O
in the environment.
-carotene,
lutein, zeaxanthin,
-cryptoxanthin, and canthaxanthin (7) . Recently, a sexual cycle was described for Phaffia(8) , which should enable biochemical studies
to be complemented by genetic analysis of carotenoid function. In
earlier reports we demonstrated that carotenoids in P. rhodozyma confer resistance to activated oxygen species. The present study
shows that certain oxygen species also regulate carotenoid levels by
induction of synthesis and oxidative degradation of pre-existing pools
of carotenoids, and through degradation of end products they relieve
feedback inhibition of the astaxanthin pathway.
Chemicals
Hydrogen peroxide (30% solution, v/v), t-butylhydroperoxide (tBOOH), dimethyl sulfoxide, potassium
hydrogen phthalate, rose bengal (RB), mevalonolactone and thymol
were from Sigma. Petroleum ether, acetone, acetic anhydride, sodium
borohydride, potassium cyanide, and -terthienyl (
-T) were
from Aldrich. Ethanol (200 proof; USP grade) was a product of Aaper
Alcohol and Chemical Co., Shelbyville, KY. Ecolume liquid
scintillation mixture was from ICN Radiochemicals (Costa Mesa, CA). (RS)-[2-
C]Mevalonolactone (55
mCi/mmol) was from Amersham Corp. or from American Radiolabeled
Chemicals Inc. (St. Louis, MO). All other chemicals used were of
analytical grade or the highest grade commercially available.
Yeast Strains and Growth
P. rhodozyma strains 67-385 and 2A2N were described
previously(9, 10) . Yeasts were maintained on slants
of yeast extract/malt extract/peptone/glucose medium (YM broth, Difco)
with 2% agar (YM agar) and refrigerated at 4 °C. Yeast strains were
also stored in 40% glycerol/60% YM broth at -70 °C.2 mg/ml was obtained. These yeasts were
then inoculated at 1% (v/v) into 30 ml of YM broth in 300-ml baffled
shake flasks and were grown for 2-5 days depending on the
experiment. Yeasts were harvested by centrifugation, and dry cell
weight, yeast viability, total carotenoid, and individual carotenoids
were analyzed as described previously(11) . To determine total
C incorporation into carotenoids, carotenoids were
extracted and purified as described previously (11) and
quantitated by label incorporation. Additionally,
-carotene
and
-zeacarotene were isolated by a second TLC run on silica gel
using 5% toluene/95% petroleum ether as the mobile phase. After
purification, individual bands were scraped and each band reeluted
three times with 2 ml of acetone. The pooled acetone extracts were
dried inside the glass scintillation vials and dissolved in 10 ml of
Ecolume liquid scintillation mixture. The vials were counted in a
Beckman model 5801 liquid scintillation counter (Irvine, CA).
Treatment of P. rhodozyma with the Peroxyl Radical
Generators H
For exposure of P.
rhodozyma to peroxyl radicals, cells in YM broth were harvested by
centrifugation at 4 °C, washed twice in 0.1 M potassium
phthalate buffer (pH 5.0), resuspended in an equal volume of buffer,
and exposed to HO
, tBOOH, and Chemically
Generated
O
O
or tBOOH. A cell
concentration of approximately 5-7 mg ml
was
used in each experiment. Higher concentrations tended to reduce the
reactions of carotenoids, while lower densities tended to cause
excessive bleaching.
C]mevalonolactone (MVA) after 36 h
growth in YM broth. The cells were harvested and washed twice in Yeast
Nitrogen base (YN; Difco Company, Detroit MI) broth buffered with 0.1 N potassium phthalate (pH 5). Cultures were then resuspended
at a cell density of 5-7 mg/ml in buffered YN broth, 30 ml was
placed in 300-ml baffled shake flasks, and 300 µl of
[2-
C]MVA (1 M MVA; 10 µCi/ml) was
added. The sodium salt of mevalonolactone was prepared immediately
prior to use by dissolving 160 mg of mevalonolactone in 600 µl of 2 N NaOH. Two hundred µl of
[2-
C]mevalonolactone (50 µCi/ml) was added
and the solution heated to 60 °C for 2 h. The solution was
neutralized with HCl and the volume brought to 1 ml.
O
produced
by photoactivation of RB or
-T. RB was prepared and used as
described previously(6) . Illumination of RB or
-T with
550 or 366 nm light, respectively, produces
O
by photocatalyzed conversion of ground state triplet oxygen (
O
) to
O
(12, 13) . For
-T
treatment,
-T was dissolved in ethanol as a 1000-fold stock
solution. The culture inoculum (10%, v/v) was exposed to
-T and
366 nm light for 5 min as described previously(6) . Inoculum
samples were plated before and after treatment with
-T and UV
light to determine the degree of inactivation of P. rhodozyma by the treatment. Cells were then cultured in the dark and sampled
over 4 days. After culturing, yeasts were harvested by centrifugation
and dry cell weight and carotenoids analyzed.
Identification of Unknown Carotenoid Formed by Exposure
of the Yeast to tBOOH or H
A
previously unreported carotenoid was detected in minor quantities in
cells exposed to tBOOH or HO
O
. Repeated
incubations of several batches of cells with H
O
were carried out to obtain sufficient pigment for analysis. The
carotenoid was initially purified in two chromatographic runs on
activated silica plates (Silica Gel 60, 20
20 cm, 0.25 mm
thickness, E. Merck, Darmstadt, Germany) using a solvent system of 20%
acetone/80% petroleum ether. It was further purified by two
chromatographic runs on activated silica plates using 40% acetone/60%
petroleum ether as the solvent system. The unknown was collected over
several weeks and stored at -70 °C under N
in the
dark. Acetylation, silylation, and NaBH
reduction were
performed according to Davies(14) . Mass spectrometry data were
collected in the electron impact mode on an MA-50TC ultrahigh
resolution mass spectrometer (Kratos Ltd.) and data collected on a
Kratos DS-55 data acquisition system. About 100 µg of the purified
carotenoid was used for mass spectrometry. Although unlikely, the
possibility that this carotenoid could be an artifact of isolation
cannot be entirely discounted since sufficient pigment was not
available to perform NMR and CD spectroscopy.
Statistical Validation of Results
All experiments
in this report were performed in duplicate or triplicate and replicated
at least once. The error estimates presented in the text and figures
for yeast mass and carotenoid content represent 1 standard deviation
from the mean.
Induction of Carotenogenesis by
In a previous study we showed that
astaxanthin protects P. rhodozyma from killing by O
O
generated by photoactivated RB or
-T(6) . In the present study we investigated the influence
of exposure to
O
and ROO on carotenoid
formation, degradation, and composition. Wild-type P. rhodozyma cultures were grown in flasks containing YM broth and increasing
concentrations of RB. The flasks were continuously illuminated with 550
nm light, which generates
O
(12) .
Continual incubation in the presence of 4-6 µM RB
increased total pigmentation by
40% after 24 and 48 h (Fig. 1A). Pigmentation usually increases with slow
growth rates; however, the RB-associated pigment increase was not
growth rate-associated since the dry cell weight after 24 h was
actually slightly higher in the 4 and 6 µM RB-treated
cultures compared to the untreated culture. The relatively small
incremental increase in carotenoid level may have been due to
limitations in the quanta of light reacting with RB in the turbid yeast
suspension. No significant change in the relative levels of individual
carotenoids was found by TLC analysis (data not shown). Higher RB
concentrations of 8 and 10 µM decreased pigmentation to
approximately the basal level of the control, which was caused by
killing of a proportion of the yeast population as indicated by lower
cell densities in the higher RB levels.
O
. A, RB + 550 nm light; B,
-T + 366 nm light.
O
would predispose
the cells to produce increased levels of carotenoid during subsequent
dark incubation possibly by gene activation as proposed by Hodgson and
Murillo (15) for Myxococcus. Pigmentation was
increased in P. rhodozyma when the inoculum was treated for 5
min with
-T activated by 366 nm light, followed by culturing for
up to 4 days in the dark (Fig. 1B). At
-T
concentrations greater than 10 µM, the pigment levels
decreased, which was associated with cell killing. At concentrations of
-T > 10 µM, cell survival after treatment in the
population was < 50%, while treatments with lower concentrations all
maintained at least 65% viability. Xanthophyll synthesis increased
maximally during stationary phase, the stage in the growth cycle in
which oxygen uptake is unaffected by CN
(see next
section). These results are intriguing because they show that brief
exposure of the cells in the inoculum is sufficient to increase the
levels of pigmentation during subsequent dark fermentation. The most
likely explanation for the increased levels is that carotenoid gene
expression was increased by the relatively brief pulse of
O
and expression continued throughout the
incubation.
Oxygen Utilization in P. rhodozyma Cultures
In
addition to formation of O
by light-activated
reactions,
O
and other active oxygen species
can be generated in dark biological systems by oxygen-utilizing
respiratory systems(16, 17, 18) . These
active oxygen species primarily derive from spillover of oxidative
metabolism and also from the activities of alternative pathways of
oxygen metabolism mediated by P-450 cytochromes, flavin oxygenases, and
peroxisomal metabolism(18) . Consequently, we determined
utilization of O
in wild-type P. rhodozyma cells
as a function of culture age (Fig. 2). Although total O
uptake remained relatively constant throughout growth and in
stationary phase, there was a nearly complete shift from
CN
-sensitive to CN
-insensitive
respiration in the stationary phase, supporting the importance of
alternative pathways of O
utilization in astaxanthin
biosynthesis. Carotenoids, particularly the oxygenated xanthophylls,
are synthesized at the maximum rate during the stationary phase (19) or when respiration is blocked by
antimycin(10, 20) .
Alterations in Relative Levels of Carotenoids by Exposure
to tBOOH
We considered that O
may be
chemically synthesized in the cell through the reaction of two peroxyl
radicals (16) (Reaction 1).
Reaction 1
We therefore treated the yeast with the peroxyl-radical
generator tBOOH. Unexpectedly, treatment with tBOOH caused substantial
changes in the carotenoid composition of the yeast ( Table 1and Fig. 3). Total carotenoid levels stayed relatively constant
during exposure to 8 mM tBOOH for 6 h. During this incubation
period, there was a sharp decrease in astaxanthin concentration and a
4-fold increase in the concentration of -carotene (Fig. 3).
Examination of the individual carotenoids in the cells by TLC also
showed the presence of a previously unreported carotenoid in P.
rhodozyma. In addition to increase in
-carotene and decrease
in astaxanthin, some other pigments were degraded or increased. HDCO
and torulene decreased in content, while early intermediates in the
pathway such as
-carotene and echinenone increased slightly.
-Zeacarotene was also detected in small quantities, suggesting
asymmetric cyclization of the linear precursors.
Figure 3:
-Carotene and astaxanthin levels in P. rhodozyma strain 67-385 as a function of exposure time to 8
mM tBOOH.
Destruction of
carotenoids also occurred but to a lesser extent when cells were
treated with HO
(Table 2). Since both
tBOOH and carotenoids are hydrophobic, whereas H
O
is hydrophilic, lower H
O
reactivity could
have been caused by poor interaction of the reactants in the cell or by
limited diffusion of H
O
through the cytoplasmic
membrane. We showed earlier that P. rhodozyma contains a low
level of catalase compared to Saccharomyces
cerevisiae(11) . Since catalase decomposes
H
O
, whereas tBOOH is a suicide substrate for
catalase(21) , it is also possible that H
O
was partly eliminated from the cell by catalase thereby
decreasing its reactivity.
Peroxyl radicals appear to react directly with carotenoids causing degradation by free radical mechanisms(7, 22, 23, 24) . To attempt to determine if peroxyl radicals were reacting with carotenoids, an inhibitor of carotenogenesis (thymol) was added 3 h prior to treatment with tBOOH (Table 3). In the presence of thymol, significant bleaching of the cells and degradation of carotenoids occurred, while incubation with thymol alone did not bleach the carotenoids (data not shown). Cells treated with tBOOH without thymol partly replenished their complement of carotenoids by de novo synthesis during the 6-h exposure.
The decrease in
concentrations of astaxanthin and certain other carotenoids in the
presence of thymol and tBOOH suggests that carotenoids react directly
with peroxyl radical. These data suggest that peroxyl radical degrades
astaxanthin, in turn relieving feedback inhibition of carotenoid
biosynthesis. However, an alternate explanation for the increased
-carotene levels coincidental to the decrease in astaxanthin
content is that peroxyl radical inhibits or eliminates the activity of
the oxygenation enzymes that converts carotenes to xanthophylls. In
order to distinguish between these two possibilities, we fed 36-h-old
cultures with [2-
C] MVA in the presence
or absence of 8 mM tBOOH and incubated the cells for an
additional 36 h. Under oxidative stress, total MVA incorporation into
carotenoids increased
6-fold. Incorporation into
-carotene
and
-zeacarotene increased
4-fold, whereas incorporation into
astaxanthin increased over 2-fold. If peroxyl radical inhibited the
-carotene oxygenases, reduced incorporation into the xanthophylls
would be expected. However, if peroxyl radical degradation of
astaxanthin relieved feedback inhibition, then MVA incorporation would
be expected to increase as the biosynthetic pathway becomes more active (Table 4). Similar results were seen after 12 h of exposure to
labeled MVA (data not shown). These results support the hypothesis of
feedback regulation by astaxanthin in P. rhodozyma.
The
unknown pigment was purified and subjected to a number of
derivitization procedures (acetylation, silylation, and NaBH reduction). The compound and the resulting derivatives were
subjected to measurements of polarity, as judged by silica gel TLC
mobility, visible light spectra, and low resolution electron impact
mass spectroscopy. From these analyses, it was determined that the
carotenoid's molecular formula was
C
H
O
. This xanthophyll contains a
chromophore of 14 conjugated double bonds. It has one cyclized
ring containing a ketone at the 4-position and a hydroxyl at the
3-position. The other end group is in the
configuration
containing a hydroxyl group at the 1`-position. Taken together, these
data indicate that the pigment is identical or very similar to
dehydroflexixanthin
(3,1`-dihydroxy-2,3,3`,4`-tetradehydro-1`,2`-dihydro-
,
-carotene-4-one).
This is only a tentative structure. Because of the small amounts
isolated (<100 µg), it was not possible to perform NMR and CD
spectroscopic analyses to unequivocally identify the carotenoid.
Active oxygen species including O,
HO
, OH, and
O
have been
suggested as biological reactants that delay or prevent degenerative
diseases such as cancer, arteriosclerosis, cataracts, and increase
longevity in eukaryotes(18, 27, 28) .
Oxidative damage to DNA, proteins, and other cellular components
accumulated with age and decreased the life span in model eukaryotes
such as Drosophila. The source of these oxidant by-products in
cells is probably excessive oxidative metabolism through respiration,
oxidative bursts of phagocytic cells, peroxisomes, cytochrome P-450
systems, and flavin-containing oxygenases(18) . Metabolism in P. rhodozyma associated with astaxanthin biosynthesis is
extremely oxygen-demanding. A mathematical model indicates that 21 mol
of O
are required per mol of astaxanthin
synthesized(29) . We have often noticed in flask and fermentor
cultures of P. rhodozyma that carotenoid levels, particularly
the xanthophylls, accumulate at a maximum rate in older cultures when
growth has terminated and O
uptake is resistant to
CN
or to antimycin. Likewise, carotenoid
concentrations increase when the yeast is grown on non-fermentable
carbon sources or in the presence of
antimycin(10, 20, 30) , conditions that may
require oxygen utilizing pathways other than mitochondrial respiration.
Several studies have implied that carotenoids have an important role
as antioxidants in P.
rhodozyma(6, 9, 11) . We demonstrated
earlier that astaxanthin provides a defense against killing by O
(6) , and this defense is probably
important in the yeast's natural habitat. It appears that
O
may be important in contributing to death of
some fungal populations in certain habitats. This is particularly true
of organisms exposed to light, such as in the phylloplane and in fluxes
of trees, and that some biochemical pathways such as carotenoid
biosynthesis may have evolved in part to protect against damaging
actions of
O
.
Although well studied in
photosynthetic systems, the roles of O
in cell
metabolism and longevity in nonphotosynthetic microorganisms and
mammalian cells have not been investigated to the same extent as other
oxygen species such as H
O
, peroxyl radicals,
and O. Singlet oxygen is known to occur as a result of respiratory
burst in mammalian cells, by transfer of energy from light in plants,
and through lipid peroxidation. Other mechanisms of dark
O
formation in chemical and biological systems
have recently been demonstrated, including generation through the
Haber-Weiss reaction (17) and on acidification of aqueous
hypochlorite(31) . Krinsky (16) hypothesized several
years ago that O, H
O
, and peroxyl radicals may
spontaneously form
O
in the cell.
Our data
suggest that O
may induce carotenoid synthesis
in P. rhodozyma by gene activation. A brief pulse of exposure
in the inoculum yielded a yeast population that produced more
astaxanthin in a dark fermentation. Hence, continual pressure by
O
was not necessary to stimulate
carotenogenesis. Hodgson and Murillo (26) presented an
interesting model of regulation of carotenogenesis in Myxococcus
xanthus, in which
O
was produced at the
cell membrane by reaction of blue light and O
with
protoporphyrin IX induced carotenogenesis. Induction of carotenogenesis
by blue light in P. rhodozyma has been reported previously (20, 32) . Induction by light may involve an
O
intermediate. The direct relation of
O
to photoregulation of carotenoid synthesis in
other organisms has not been studied to our knowledge. However,
photoregulation has been shown to affect the production of
carotenogenic enzymes in certain organisms. Fontes et al. (33) showed that the carR gene, encoding phytoene
dehydrogenase, was induced by light. Light also induced the formation
of hydroxymethylglutaryl-CoA reductases in the carotenogenic yeast Rhodotorula minuta(34) . We detected no significant
change in the relative amounts of the various carotenoids after
O
exposure, indicating that
O
induction in P. rhodozyma occurs early in the
biosynthetic pathway and possibly affects regulatory genes or proteins.
We also noted that peroxyl radicals generated by tBOOH dramatically
affected carotenoid levels and composition in P. rhodozyma.
Peroxyl radical can react directly with carotenoids as well as many
other molecules in cells, and may form O
under
specific conditions. Peroxyl radicals, because of their relatively long
life in the cell, their ability to diffuse, and their selectivity, are
potentially more harmful than other types of radicals (7, 35) . Certain carotenoids are very effective
quenchers of peroxyl radicals(7, 23) . Our data
support the hypothesis that carotenoids react in vivo with
peroxyl radicals. Exposure of P. rhodozyma to tBOOH or
H
O
markedly altered the relative concentrations
of the carotenoid composition. In particular, astaxanthin was destroyed
and the concentration of carotenes, particularly
-carotene, were
increased. Since the carotene pool was increased as the astaxanthin
concentration diminished, it is possible that astaxanthin regulates
carotenoid biosynthesis by feedback inhibition of carotene synthesis
early in the pathway. The addition of the biosynthesis inhibitor thymol
together with tBOOH resulted in rapid destruction of the carotenoids
within minutes, suggesting that there is a rapid turnover of
carotenoids in P. rhodozyma. These results imply that
astaxanthin biosynthesis involves a kinetically active and yet highly
regulated and dynamic system involving induction (by
O
), degradation of xanthophylls by peroxyl
radicals, and rapid replenishment of carotenoid pools when the
xanthophylls are destroyed.
Incubation of yeasts with tBOOH and
HO
also resulted in the production of a new
carotenoid, tentatively identified as dehydroflexixanthin, which may
actually be a carotenoid breakdown product. The formation of this
product was rapid, occurring in less than 15 min, and did not require
new protein synthesis. However, it was produced only in live (unheated)
cells, implying degradation entails an in vivo cellular
response to the presence of peroxyl radicals and is catalyzed by
specific enzymes.
The actual biosynthetic pathway to astaxanthin in P. rhodozyma is enigmatic. An (9) proposed a primary
and an alternate biosynthetic pathway for astaxanthin. This was based
on the detection of carotenoids that do not logically fit into the
pathway proposed initially by Andrewes et al.(26) .
However, this proposal did not take into account the possibility that
some of the carotenoids detected in P. rhodozyma may be
enzymatically derived oxidative products formed in reactions with
radicals. The actual astaxanthin biosynthetic pathway in P.
rhodozyma may indeed be the one originally proposed by Andrewes et al.(26) ; however, at the time he also could not
account for the presence of HDCO
(3-hydroxy-3`,4`-didehydro-,
-carotene-4-one) in substantial
cellular quantities. This pigment may also be an enzymatic degradation
product of another xanthophyll. This hypothesis is currently being
investigated in our laboratory. The occurrence of asymmetric pigments
may be due to the activity of a bifunctional enzyme complex that
catalyzes reactions on both ends of the fairly symmetrical carotene
precursors. Under stress, catalysis is disrupted and asymmetric
products are produced which may be further modified by oxidative and
degradative reactions.
In summary, our results suggest that
induction by O
, end product inhibition by
astaxanthin, and the activity of degradative pathways in concert
control the levels of carotenoids in P. rhodozyma. The
distinct differences in carotenoid metabolism mediated by
O
and peroxyl radicals can be explained by
their differing interactions with carotenoids. Singlet oxygen is
detoxified by carotenoids through changes in energy state but without
disruption of covalent structure of the carotenoids. In contrast,
carotenoids covalently react with peroxyl radicals and then are further
degraded, possibly by reactions involving cytochrome P-450 or
flavin-oxygenases as occurs with other terpenoids and steroids. The
astaxanthin pathway in P. rhodozyma appears to involve a
dynamic system in which carotenoids are in a constant state of
synthesis and degradation. Both the synthetic and degradative systems
appear to respond to oxidative stress very rapidly. Exposure to
O
can alter the total levels of carotenoid
intermediates in minutes to hours, and distinct degradative products
are also formed rapidly in response to peroxyl radicals. P.
rhodozyma appears to have evolved a sophisticated system with the
ability to rapidly alter its carotenogenic capacity depending on both
environmental conditions as well as the physiological state of the
cell.