(Received for publication, February 6, 1996, and in revised form, December 19, 1996)
From the Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., Kanazawa-ku, Yokohoma-shi, Kanagawa 236, Japan
Escherichia coli strains expressing
the marine bacteria (Agrobacterium aurantiacum and
Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes
(crtZ and W), Haematococcus pluvialis
bkt, and Erwinia uredovora crtZ genes were used for
in vitro characterization of the respective enzymes.
Specific enzyme assays indicated that all of the enzymes are
bifunctional, in that the CrtZ enzymes formed zeaxanthin from
-carotene via
-cryptoxanthin, as well as astaxanthin from
canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes
synthesized canthaxanthin via echinenone from
-carotene and
4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from
zeaxanthin. Comparison of maximum catalytic activities as well as
selectivity experiments carried out in the presence of both utilizable
substrates indicated that the CrtZ enzymes from marine bacteria
converted canthaxanthin to astaxanthin preferentially, whereas the
Erwinia CrtZ possessed a favorability to the formation of
zeaxanthin from
-carotene. The CrtW/BKT enzymes were not so defined
in their substrate preference, responding readily to fluctuations in
substrate levels. Other properties obtained indicated that the enzymes
were strictly oxygen-requiring; and a cofactor mixture of
2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to
activity. Based on enzymological data, a predicted pathway for
astaxanthin biosynthesis is described, and it is proposed that
CrtZ-like enzymes be termed carotenoid 3,(3
)-
-ionone ring hydroxylase and CrtW/BKT carotenoid 4,(4
)-
-ionone ring
oxygenase.
Astaxanthin (3,3-dihydroxy-
,
-carotene-4,4
-dione) is the
most commonly found carotenoid pigment in marine animals (1). It is
responsible for the red/pink coloration of crustaceans (1), shellfish
(2), and the flesh of salmonoids (3). Despite high endogenous levels,
these marine animals do not possess the ability to synthesize
astaxanthin or other carotenoid pigments de novo. Instead
carotenoid pigments must be acquired via their diet (4). Industrially,
astaxanthin has been exploited as a feed supplement for cultured fish
and shellfish (5-7). Other diverse biological functions of astaxanthin
include an involvement in cancer prevention (8), enhancer of immune
responses (9), and a free radical quencher (10, 11). It is evident,
therefore, that astaxanthin is a molecule with potential both to the
pharmaceutical and food industries.
Astaxanthin belongs to the class of compounds known as xanthophylls.
These are carotenoids modified with oxygen-containing functional
groups. Xanthophylls are found universally in chloroplast-containing plant tissues. However, the biosynthesis of astaxanthin is limited in a
virtually exclusive manner to microorganisms, for example, the yeast
Phaffia rhodozyma (12), the freshwater alga
Haematococcus pluvialis (13) and the marine bacteria
Agrobacterium aurantiacum and Alcaligenes sp.
strain PC-1 (14). Recently the genes involved in the formation of
astaxanthin and its intermediates have been isolated and functionally
characterized in vivo by complementation, providing the
first insight into the biosynthetic route (15). The pathway proposed
(Fig. 1) is based on the lack of enzyme specificity surmised from complementation studies (15). The hydroxylation of
-carotene at positions 3 and 3
on the
-ionone ring forming zeaxanthin via
-cryptoxanthin is mediated by the product of the gene
designated crtZ, which has been isolated from
Erwinia species (16, 17) and marine bacteria (15). The
direct conversion of methylene to keto groups at positions 4 and 4
on
the
-ionone ring forming canthaxanthin via echinenone are reactions
performed by the gene product encoded by the crtW gene from
marine bacteria (18) and bkt gene of H. pluvialis
(19). A comparison of the deduced amino acid sequences indicates the
existence of 90% identity between the marine bacteria A. aurantiacum and Alcaligenes PC-1 CrtZ, which in turn
show 54% identity with the Erwinia species. The CrtW
proteins possess a 75% identity among the marine bacteria species,
whereas a 37% identity is evident when compared with the BKT gene
product of H. pluvialis. When combinations of the gene
products responsible for the introduction of the hydroxyl moieties at
positions 3,3
and formation of keto groups at 4,4
positions on the
-ionone ring are expressed in Escherichia coli, astaxanthin as well as substantial quantities of various intermediates are synthesized (15). Because of the limitations of in vivo complementation, the bifunctional character of the CrtZ and/or CrtW
type gene products has not been ascertained conclusively. Thus it
remains unclear whether CrtZ can convert canthaxanthin to astaxanthin
via phoenicoxanthin (adonirubin) or CrtW can convert zeaxanthin to
astaxanthin via 4-ketozeaxanthin (adonixanthin).
At present no specific in vitro assay systems, cofactor requirements, or properties of the enzymes originating from an astaxanthin-forming organism have been reported. Advances in carotenoid enzymology have, however, been hindered by the practical difficulties associated with their assay (20, 21). The heterologous expression of the recently isolated astaxanthin biosynthetic genes in E. coli has provided a valuable opportunity to study the respective enzymes. In this article the in vitro characterization of the marine bacteria CrtZ and CrtW enzymes specific to astaxanthin biosynthesis are reported for the first time as well as a comparison with the Erwinia CrtZ and Haematococcus BKT.
Plasmids pCAR16crtX and pCAR25
crtX,
corresponding to pCAR16delB and pCAR25delB (16), contain the
Erwinia uredovora genes for the synthesis of
-carotene
and zeaxanthin, respectively. pACCAR16
crtX has been described (18).
pCRT-Z containing the E. uredovora crtZ gene was constructed
by inserting the SphI(5599)-EcoRI(6505) fragment
of pCAR25 (16) into the EcoRI-SphI site of pUC18.
Plasmids pAK916 and pAK96NK, which contain the A. aurantiacum
crtW and crtZ genes, respectively, were described
previously (15). pPC17-3 (18) and pPC13 (15) carried the
Alcaligenes sp. strain PC-1 crtW and
crtZ genes, respectively. Plasmid pUCBKT containing the H. pluvialis bkt gene was constructed by polymerase chain
reaction using plasmid pHP51 (19) as a template, where ATG at
nucleotide position 264 (19) was placed next to the HindIII
site of pUC19 to form a fusion protein with the amino terminus of
-glucosidase as follows.
![]() |
![]() |
![]() |
The E. coli strains were grown in 2 × YT medium (22) containing the
appropriate additions as described below. In all cases an initial
overnight culture (5 ml) was prepared from a glycerol stock. An aliquot
constituting 1% by volume of the fresh culture medium was used to
inoculate cultures for induction. These cultures contained 0.1 mM isopropyl 1-thio--D-galactopyranoside and
the following antibiotics depending on the recombinant strain. Strains used and antibiotic requirements follow. E. coli JM109
containing plasmids pCAR16
crtX, pCAR25
crtX, pCRT-Z, pAK916,
pAK96NK, pPC17-3, pPC13, and pUCBKT required ampicillin at a final
concentration of 150 µg/ml. E. coli JM101 containing
pACCAR16
crtX and pAK916, which synthesized canthaxanthin (18),
required ampicillin (150 µg/ml) and chloramphenicol (30 µg/ml). All
E. coli strains were grown at 30 °C and shaken at 200 rpm. Marine bacteria A. aurantiacum and
Alcaligenes PC-1 were grown in Sakagachi flasks (Eagleglass, Tokyo) containing 150 ml of marine broth (Difco). Culture vessels were
shaken (164 rpm) at 20 °C for 4 days under diffuse light.
Recombinant E. coli extracts were prepared from cells induced for 4 or 6 h. Cells were collected by centrifugation at 6,000 × g. Pelleted cells were placed on ice and resuspended in 100 mM Tris-HCl, pH 8.0, containing 1 mM dithiothreitol, protease mixture (0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin) as well as 5% glycerol v/v. The suspension was passed through a French press cell at a internal pressure of 500 p.s.i. DNase (50 µg) was added to the broken extract, and the mixture was incubated on ice for 30 min. The crude extract was centrifuged at 10,000 × g to remove cell debris. When determining cofactor requirements the supernatant was desalted with a PD10 column (Pharmacia Biotech Inc.) in 100 mM Tris-HCl, pH 8.0, containing 1 mM dithiothreitol.
Incubation ConditionsIncubations were carried out in a
total volume of 800 µl. Typically 400 µl of the substrate extract
of E. coli producing -carotene, canthaxanthin, or
zeaxanthin was added (the precise volume varied depending on the
quantity of substrate supplied). An equal volume of the E. coli extract to be analyzed was also added. The remaining 200 µl
was buffered with 0.4 M Tris-HCl, pH 8.0, containing 1 mM dithiothreitol, 0.1% v/v Tween 60, 3 mM
ATP, 0.5 mM FeSO4, 0.5 mM
2-oxoglutarate, 5 mM ascorbic acid. The presence of 1 mg/ml catalase was optional. The mixture was incubated by shaking in the dark
for 4 h at 30 °C. When performing incubations with both utilizable substrates the mixture could be boiled for 30 s to remove any endogenous activities. Incubations were terminated by the
addition of methanol and stored at
70 °C under an atmosphere of
nitrogen. Anaerobic incubations were performed in Thunberg tubes (23).
Extracts and incubation mixture were degassed under vacuum then purged
with nitrogen gas prior to sealing. Control incubations were performed
simultaneously in all experiments. They were identical apart from the
presence of an E. coli extract from a non-crtZ or
crtW expressing strain, as well as incubations containing
heat-denatured (boiled 1 min) enzyme preparations.
Carotenoid standards were extracted and purified from recombinant E. coli strains and A. aurantiacum using general carotenoid methodology as described (24). Products and substrates from the in vitro incubations were extracted with 10% (v/v) diethyl ether in petroleum ether 40-60 °C (3 volumes). After mixing, a partition was formed by centrifugation at 3,000 × g for 5 min at 4 °C. The organic phase was removed and aqueous phase reextracted with the same solvent. The remaining aqueous phase was further reextracted with chloroform (2 volumes). The organic extracts were pooled and brought to dryness under a stream of nitrogen.
Carotenoids formed in vitro were separated by
HPLC1 on a reversed phase C18
column (Nova-pak HR 6 µ C18, 3.9 × 300 mm, Waters) using an isocratic mobile phase of acetonitrile/methanol/isopropyl alcohol (90:6:40, v/v/v). On-line photodiode array detection enabled peak identification by comparison of spectral data with authentic standards (24). Retention times and max, respectively,
are as follows: astaxanthin, 5.31 min, 480 nm; 4-ketozeaxanthin, 5.96 min, 465 nm; phenicoxanthin, 6.61 min, 475 nm; zeaxanthin, 7.09 min,
456 nm; canthaxanthin, 9.05 min, 475 nm;
-cryptoxanthin, 20.3 min,
452 nm; echinenone, 22 min, 460 nm;
-carotene 60.6 min, 456 nm.
Quantification of carotenoids was achieved by injecting known amounts
of carotenoid standards to generate a standard curve. In addition,
further confirmation of the amount eluting and detected on-line was
performed using the method devised by Sandmann (35). The detection
limit of HPLC-photodiode array detection system was 0.0003 absorbance
units corresponding to 0.5 pmol/h/mg of protein enzyme activity. Below
this level novel peaks were detectable having the correct retention
time, but definitive spectral data were difficult to derive and
therefore are referred to as trace levels.
Protein present in E. coli extracts was estimated using the Bio-Rad protein assay dye reagent protocol following the manufacturer's instructions. Enzyme activity was expressed as pmol formed per h/mg of protein in all cases. When determining the in vivo rate of carotenoid formation carotenoid-synthesizing E. coli cultures were induced and time points taken at 1-h intervals. In vivo and in vitro comparisons were made between cells induced for the same time period in the case of recombinant strains.
Substrates were supplied in the form of extracts
prepared from recombinant E. coli strains accumulating
-carotene, zeaxanthin, and canthaxanthin in the all-trans
configuration. This procedure overcame difficulties experienced when
introducing carotenoids into aqueous incubation mixtures. All varieties
of the CrtZ enzymes formed zeaxanthin from
-carotene via the
monohydroxylated compound
-cryptoxanthin. The intermediate
-cryptoxanthin was detectable at significant levels (20-40%) as
shown in Fig. 2A. When canthaxanthin was
supplied to the CrtZ enzymes astaxanthin was formed via phoenicoxanthin in all cases. Astaxanthin was the predominant product constituting (>70%) of the total products (Fig. 2B). Canthaxanthin was
the predominant (63-68%) product synthesized by CrtW/BKT from
-carotene; significant amounts (30-35%) of its mono-keto
intermediate echinenone were also detected. The ratio of canthaxanthin
to echinenone was independent of species (Fig. 2C). When
supplied with zeaxanthin, CrtW and BKT preparations only formed trace
levels (0.5 pmol/h/mg of protein) of astaxanthin, but 4-ketozeaxanthin
was formed predominantly by the enzyme from all varieties (Fig.
2D). Why the CrtW and BKT enzymes were unable to catalyze
the conversion of 4-ketozeaxanthin to astaxanthin from the initial
substrate zeaxanthin efficiently is difficult to explain. It is
possible that the experimental conditions were not optimal. However,
in vivo 4-ketozeaxanthin can accumulate (up to 50% of the
total carotenoid content) in recombinant E. coli strains
forming ketocarotenoids (15), and in the marine bacteria it is often
more predominant than astaxanthin (14). Such findings correlate with
the in vitro data reported in this study and could suggest
that the formation of the 4
keto group on 3,3
-hydroxylated
-ionone
ring-containing carotenoids is prevented if prior keto group formation
at position 4 has occurred.
Reaction products were separated by HPLC and identified with an on-line
photodiode array detector, from which spectral data could be derived
(described under "Experimental Procedures"). Comparison with
authentic standards enabled conclusive identification by
cochromatography and comparative spectral data. In all cases the CrtZ/W
and BKT reaction products were of the all-trans
configuration and constituted novel compounds not present in control
samples. Controls were performed with both extracts of E. coli strains not harboring astaxanthin-forming genes and
heat-denatured extracts. Some compounds present at low levels did occur
in the controls and were also detectable in the experimental
incubations, but they did not cochromatogram with the CrtZ/W and BKT
reaction products and were spectrally unrelated. Presumably these
compounds were derived from the substrates due to nonspecific
oxidation. Thus it can be confirmed that the enzymes CrtZ/W of marine
bacteria, CrtZ of Erwinia, and BKT of
Haematococcus are bifunctional in their activity.
Hydroxylation of carotenoid -ionone rings by CrtZ can be performed
specifically at the 3,3
positions regardless of the presence of
previous keto group formation at positions 4,4
. Although the formation
of keto groups by CrtW/BKT is specific at positions 4,4
on the
-ionone rings retrospective of previous 3,3
hydroxylation, it must
be stated that the mono-keto reaction product accumulates if prior 3,3
hydroxylation has occurred. In this study, we have only used
carotenoids containing
-ionone rings as they are the precursors
involved in astaxanthin formation. It would be interesting to ascertain
whether the substrate specificity and product diversity would extend to
-ring cyclic carotenoids or even acyclic carotenoids.
In Table I the maximum catalytic activities are
presented. These data indicate that compared with the
Erwinia CrtZ the marine bacteria enzymes possess greater
(e.g. A. aurantiacum, 10-fold) ability to convert
canthaxanthin to astaxanthin. In contrast, the Erwinia
enzyme shows greater (e.g. 10-fold compared with
Alcaligenes) activity for the conversion of -carotene to
zeaxanthin than canthaxanthin to astaxanthin. Of the CrtZ enzymes the
marine bacterium A. aurantiacum possessed the greatest
activity. Comparison of CrtW/BKT activities using
-carotene as
substrate indicated that the Alcaligenes PC-1 CrtW was about
2-fold greater than the other varieties which were similar in activity
(Table I). 4-Ketozeaxanthin was formed from zeaxanthin at a similar
rate by all of the enzymes examined.
|
The in vivo rate of carotenoid formation in recombinant strains synthesizing zeaxanthin (CrtZ), canthaxanthin (CrtW), and astaxanthin (CrtZ+ W/BKT) as well as the marine bacteria were determined as 5.3 ± 1.9, 1.8 ± 0.2, 2.95 ± 0.95, and 3.1 ± 0.7 µg/g dry weight/h, respectively. Corresponding to typical in vitro rates for CrtZ enzymes of 2.5 µg/g dry weight/h (mean from typical experiment) and CrtW/BKT 0.83 µg/g dry weight/h (mean from typical experiment). The in vitro rates for CrtZ and W/BKT, respectively, are 47 and 46% of the in vivo rate. To our knowledge no previous data have been reported comparing in vivo and vitro rates of carotenoid formation. Other enzymes involved in terpenoid biosynthesis have been compared with their in vivo biosynthetic capacity. In these instances it was found that the enzymes camphor hydroxylase, abietadiene and abietadienol hydroxylase only constituted 10-20% (25) or 1% (26) of the in vivo capacity. Considering that carotenoids like other terpenoids are hydrophobic (preventing effective use in aqueous solution), easily degraded, oxidized, and prone to isomerization, it is not surprising that in vitro rates are typically a poor reflection of their in vivo counterparts. Taking these facts into account along with the strong analogy between qualitative product formation in vitro and in vivo, we believe it is justified that at present the in vitro system described represents a creditable representation of the in vivo situation.
Oxygen Dependence of CrtZ- and W/BKT-catalyzed ReactionsThe
absence of oxygen on carotenoid hydroxylation and oxygenation in
vitro was assessed. Table II shows experimental
data obtained with the A. aurantiacum enzymes using the
substrates -carotene, canthaxanthin, and zeaxanthin independently.
It was evident that all catalyzed reactions studied were
oxygen-dependent, each having a similar degree of
sensitivity to the absence of oxygen. Phoenicoxanthin was formed at a
rate of 17.0 pmol/h/mg of protein, quantitatively similar to the
aerobic rate of 22.0 pmol/h/mg of protein. However, when the levels of
phoenicoxanthin formed aerobically and aerobic are compared in a
qualitative manner with respect to their proportion of total CrtZ
products it is significant that phoenicoxanthin the monohydroxylated
product is the predominant product compared with astaxanthin
(dihydroxylated) under aerobic conditions. When the aerobic CrtZ
activity was reduced to a total rate quantitatively similar to the
anaerobic level, qualitatively astaxanthin remained the principal
product. Thus, predominance of phoenicoxanthin in the anaerobic
incubation was a finding specific to those conditions. Similar findings
of mono-ketolated product accumulation were observed with CrtW and
-carotene as substrate. It was impossible to observe any difference
in the product profile formed from zeaxanthin by CrtW/BKT as the
activity was not detectable, and as stated previously 4-ketozeaxanthin is the principal product of this reaction. From the experimental data
it can be concluded that oxygen is involved in all of the reactions
performed by the CrtZ and W enzymes from marine bacteria; this also
reflects the situation found with BKT and Erwinia CrtZ (data
not shown). At present we cannot conclusively ascertain whether the
oxygen moieties present in the carotenoid molecule resulting from
enzymatic synthesis are derived directly from molecular oxygen or if
oxygen is indirectly utilized in the catalysis. These findings are the
first in vitro evidence that the enzymes involved in
astaxanthin biosynthesis are oxygen-dependent, and they
complement previous in vivo studies described for zeaxanthin
formation by Flavobacterium species (27), xanthophyll
synthesis in leaf tissue (28), astaxanthin accumulation in P. rhodozyma (29), and xanthophyll formation in marine bacteria (34).
The changing pattern of products formed with reduced oxygen levels
suggests that its presence or absence could also play a crucial
regulatory role over product formation and could possibly be influenced
by environmental conditions.
|
In the absence of any exogenously added cofactors significant activity (20%) was detectable. Therefore there was no absolute requirement for cofactors using these enzyme preparations. The addition of the dinucleotides NAD+, NADP+ and flavins FAD, FMN (at a concentration of 1 mM) as well as divalent ions Mg2+ and Mn2+ to the incubations individually and in combination had no stimulatory effect. In fact they were often inhibitory. The presence of NADPH+ (1 mM) did yield some elevation (2.5-fold) in activity. These findings were consistent among the marine bacteria CrtZ/W, Erwinia CrtZ and BKT enzyme.
Combinations of cofactors used for typical monooxygenase (30) or
dioxygenase (31) reactions were tested subsequently. The monooxygenase
cofactors consisted of FAD, NADPH+, and ATP. This cofactor
combination increased the activity of CrtW and Z about 2.5- and
1.5-fold greater than the control, respectively. When the dioxygenase
mixture (Fe2+, 2-oxoglutarate, ascorbic acid, and catalase)
was used, far greater stimulation in activity was found, particularly
in the case of CrtZ, where about a 6-fold elevation was experienced.
CrtW activity was also increased significantly (4-fold). Based on the
observed stimulation in activities, a detailed analysis of the
components was made. A similar profile in activities was observed
between the CrtZ/W-catalyzed reactions as shown in Table
III. The presence of Fe2+, 2-oxoglutarate,
and ascorbic acid in combination was responsible for the greatest
enzymatic activity; however, all incubations containing
Fe2+ possessed an activity higher than the control value,
indicating that Fe2+ was the most influential effector. A
series of control experiments confirmed that this cofactor mixture
could not perform these conversions without the presence of the
enzymes. Their presence enhanced specific 3,3 hydroxylation by CrtZ
or 4,4
oxygenation by CrtW. Comparison between the gene sequences
encoding the marine bacteria CrtZ and W, E. uredovora and
Erwinia herbicola as well as bkt revealed a
number of conserved histidine residues in a formation consistent with
reported Fe2+ binding motifs (HX3H,
HX2HH, or HX2HH). Such motifs are
characteristic of enzymes containing non-heme iron such as
membrane-bound fatty acid desaturases (32). Fig. 3 shows
the alignment of the postulated Fe2+ binding motifs present
in the CrtZ genes from marine bacteria and Erwinia species.
Three conserved Fe2+ motif regions exist occurring at
H25X4H, H38X2HH, and
H123X2HH. In the case of BKT and the CrtW
enzymes of marine bacteria such motifs occur at
H130X3H, H168X2HH as
well as an overlapping conserved sequence between
H289X3H and H294X2HH.
Thus both biochemical and molecular data suggest that Fe2+
is a cofactor involved in the catalysis performed by these enzymes. Of
the other cofactors (2-oxoglutarate and ascorbic acid) present in the
optimal combination no independent stimulatory role was witnessed.
Perhaps their function may be as oxidizable cosubstrates, which
generate a reactive ferryl ion in the process. Such a mechanism would
be similar to the gibberellin oxidases and hydroxylases (31). However,
in the experimental system described in this article it is likely that
ascorbic acid is substituting for a genuine cosubstrate as ascorbic
acid is not present endogenously in E. coli. Purification of
these enzymes will hopefully enable determination of the precise role
performed by these postulated cofactors as well as in vitro
mutagenesis of the postulated Fe2+ binding sites.
|
Selectivity of Substrate Utilization by CRTW/Z
To investigate
the preference of one substrate above the other by these bifunctional
enzymes experiments were designed whereby both utilizable substrates
were added to the enzyme preparation simultaneously, initially in an
equal ratio. The resulting activities were calculated for the
individual reactions rates then expressed as the percent of the total
activity. The marine bacteria CrtZ enzymes showed that canthaxanthin
was the favored substrate for conversion, 36-fold in the case of
A. aurantiacum and 6-fold in the case of
Alcaligenes PC-1. The E. uredovora CrtZ in
contrast showed a 5-fold preference for -carotene as the substrate
to zeaxanthin (Fig. 4A). The CrtW enzymes and
BKT gene product were less conclusive in their selectivity with both
BKT and A. aurantiacum possessing about a 2-fold preference
for zeaxanthin as the substrate for 4-ketozeaxanthin formation.
Alcaligenes CrtW, however, showed no significant
favorability to zeaxanthin as the principal substrate for conversion
(Fig. 4B). To determine if this preference for substrates
was consistent regardless of varying substrate levels, different ratios
of substrates were incubated with the respective enzymatic
preparations; the findings are presented in Fig. 5, A and B. In the case of CrtZ from
Alcaligenes PC-1, increasing the level of
-carotene to a
level three times greater than canthaxanthin did not alter the
preference of the enzyme for the conversion of canthaxanthin to
astaxanthin (Fig. 5A). In fact it appeared to some extent
that such elevated levels of
-carotene could inhibit the conversion
of
-carotene to zeaxanthin presumably by substrate inhibition. The
A. aurantiacum CrtZ also showed no alteration in its
preference for canthaxanthin conversion even when
-carotene levels
were increased six times greater than that of the canthaxanthin substrate (Fig. 5A). In contrast, only relatively small
increases (1.5-3.0-fold) in canthaxanthin levels were required to
eliminate completely the presence of zeaxanthin formation (Fig.
5A) by the CrtZ enzymes from either marine bacteria. The
E. uredovora CrtZ was not studied extensively, but it was
evident that increases in the proportion of canthaxanthin did alter
comparatively the preference of the enzyme for the conversion of
canthaxanthin to astaxanthin to some extent (Fig. 5A).
The A. aurantiacum CrtW gene product showed a significant
response to changes in substrate concentration, as when -carotene was in excess (6-fold) canthaxanthin was the product formed. When zeaxanthin was supplied to excess (2-fold) 4-ketozeaxanthin was formed
preferentially (Fig. 5B). Comparison of the two situations suggested that the zeaxanthin to 4-ketozeaxanthin is the favored reaction as smaller incremental increases in substrate were required to
result in a greater commitment to this reaction. The
Alcaligenes PC-1 CrtW also showed an ability to respond to
changes in precursor levels, as when
-carotene was in excess
canthaxanthin was the sole product formed, and when zeaxanthin levels
were elevated 4-ketozeaxanthin resulted and canthaxanthin formation
eliminated. Although not investigated to the same extent, BKT clearly
responded to changes in precursor levels in a manner similar to that of the marine bacteria enzymes.
Collectively, data shown in Table I along with results determined in
the presence of both utilizable substrate in equal (Fig. 4) and varying
concentrations (Fig. 5), indicate that the marine bacteria CrtZ enzymes
significantly favor performing the conversion of canthaxanthin to
astaxanthin, whereas the Erwinia CrtZ has a recognizable
preference for converting -carotene to zeaxanthin. In some respects
this is not too surprising considering that the Erwinia
species accumulate zeaxanthin or its glucoside but no ketocarotenoids
endogenously. In vivo complementation studies have
established that both varieties of enzyme have the potential to perform
identical reactions (15). The CrtW/BKT enzymes are significantly less
selective in their substrate preference compared with the CrtZ enzymes.
When equal ratios of substrates are used the enzymes utilization is
similar; but compared with the CrtZ enzymes only small elevations of
zeaxanthin or
-carotene substrates are required to alter the product
profile to 4-ketozeaxanthin or canthaxanthin, respectively.
Based on the enzymes' substrate utilization we predict that the
astaxanthin pathway in marine bacteria occurs ideally through the
initial formation of canthaxanthin via echinenone, which is then acted
upon by CrtZ enzymes to form astaxanthin. This proposal is based on the
facts that -carotene is the initial precursor to oxygenated
carotenoids, and the marine bacteria CrtZ favors the conversion of
canthaxanthin to astaxanthin in vitro. However, the
bifunctional nature of the enzymes must be taken into account, and
therefore if zeaxanthin is formed initially either due to environmental
influences or in the case of recombinant studies altered expression
levels, the CrtW/BKT enzymes will utilize this as a substrate-forming
4-ketozeaxanthin, which will accumulate because of the enzyme's
apparent ineffectiveness to convert 4-ketozeaxanthin to astaxanthin. In
the recent publication by Yokoyama and Miki (33), the astaxanthin
pathway was proposed from the accumulation of intermediates
under different culture conditions. The study indicated that when
zeaxanthin was formed, substantial 4-ketozeaxanthin also
accumulated, a finding supporting the pathway deduced from the
capabilities of the enzymes determined in vitro. Other
pathways to astaxanthin via hydroxyechinenone intermediates must await further assay development before they can be confirmed. The
similarities of BKT to the CrtW enzymes would suggest that the pathway
in Haematococcus is like the marine bacteria, as suggested
in previous in vivo studies (34), but the yet to be isolated
hydroxylase gene from this organism prevents confirmation.
The in vitro characterization
of astaxanthin biosynthetic enzymes described in this article provides
the first enzymological data on their substrate utilization and product
diversity as well as possible cofactor requirements. The data presented
indicate that the CrtZ-type enzymes show properties of enzymes
frequently categorized as hydroxylases, and CrtW-type enzymes,
oxygenases. Both are also bifunctional with regard to substrates
containing -ionone rings. It is for these reasons that we propose
that the enzymes be referred to as 3,(3
)-
-ionone ring hydroxylase
and 4,(4
)-
-ionone ring oxygenase as standard enzyme
nomenclature.
We are grateful to the members of the Kirin Metabolic Engineering group and Dr. S. Ferri for helpful discussion. We also thank Dr. David E. Cane of Brown University for advice regarding possible CrtZ/W enzymatic mechanisms.