EUKARYOTIC CELL, Ap . 2007, p. 650–657 Vol. 6, No. 4
1535-9778/07/$08.00⫹0 doi:10.1128/EC.00392-06
Copy igh © 2007, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Re inal Biosyn hesis in Fungi: Cha ac e iza ion o he Ca o enoid
Oxygenase Ca X om Fusa ium ujiku oi
䌤
Al onso P ado-Cab e o,
1
Daniel Sche zinge ,
2
Ja ie A alos,
1
and Salim Al-Babili
2
*
Facul y o Biology, Albe -Ludwigs Uni e si y o F eibu g, Schaenzles . 1, D-79104 F eibu g, Ge many,
2
and
Depa amen o de Gene´ ica, Facul ad de Biologı´a, Uni e sidad de Se illa, Se ille, Spain
1
Recei ed 8 Decembe 2006/Accep ed 4 Feb ua y 2007
The ca gene clus e o he ascomyce e Fusa ium ujiku oi encodes wo enzymes esponsible o o ulene
biosyn hesis (Ca RA and Ca B), an opsin-like p o ein (Ca O), and a pu a i e ca o enoid clea ing enzyme
(Ca X). I was p esumed ha Ca X ca alyzes he o ma ion o he majo ca o enoid in F. ujiku oi, neu os-
po axan hin, a clea age p oduc o o ulene. Howe e , a ge ed dele ion o ca X did no impede neu ospo ax-
an hin biosyn hesis. On he con a y, ⌬ca X mu an s showed a signi ican inc ease in he o al ca o enoid
con en , indica ing an in ol emen o Ca X in he egula ion o he pa hway. In his wo k, we in es iga ed he
enzyma ic ac i i y o Ca X. The exp ession o he enzyme in ␤-ca o ene-accumula ing Esche ichia coli cells led
o he o ma ion o he opsin ch omopho e e inal. The iden i y o he p oduc was p o en by high-pe o mance
liquid ch oma og aphy and gas ch oma og aphy-mass spec ome y. Subsequen in i o assays wi h he e -
ologously exp essed and pu i ied Ca X con i med i s ␤-ca o ene-clea ing ac i i y and e ealed i s capabili y o
p oduce e inal also om o he subs a es, such as ␥-ca o ene, o ulene, and ␤-apo-8ⴕ-ca o enal. Ou da a
indica e ha he occu ence o a leas one ␤-ionone ing in he subs a e is equi ed o he clea age eac ion
and ha he clea age si e is de e mined by he dis ance o he ␤-ionone ing. Ca X ep esen s he i s
e inal-syn hesizing enzyme epo ed in he ungal kingdom so a . I seems likely ha he o med e inal is
in ol ed in he egula ion o he ca o enoid biosyn he ic pa hway ia a nega i e eedback mechanism.
Ca o enoids a e widesp ead lipophilic pigmen s syn hesized
by all pho osyn he ic o ganisms and some nonpho osyn he ic
ungi and bac e ia. These yellow, o ange, and ed isop enoid
compounds ul ill di e se unc ions in all axa. In addi ion,
ca o enoids a e he p ecu so s o se e al physiologically essen-
ial compounds, like he ubiqui ous ch omopho e e inal, he
phy oho mone abscisic acid (ABA), and he ungal sex ho -
mone ispo ic acid.
The syn hesis o hese ca o enoid clea age p oduc s, known
as apoca o enoids, is ca alyzed in gene al by ca o enoid oxy-
genases, which cons i u e a new nonheme i on enzyme amily
common in all axa ( o e iews, see e e ences 1, 11, 15, and
24). Recen ly, he c ys al s uc u e o a membe o his amily,
he Synechocys is apoca o enoid oxygenase, was elucida ed a
2.4-Å esolu ion. The enzyme con ains an Fe
2⫹
-4-His a ange-
men a he axis o a se en-bladed ␤-p opelle chain old co -
e ed by a dome o med by six la ge loops (20). I has also been
shown ha he 15,15⬘BCOI(␤-ca o ene oxygenase I) ac s as
a monooxygenase (21). Howe e , ecen in es iga ions o
A CCD1 (A abidopsis haliana ca o enoid clea age dioxygen-
ase 1) sugges ed a dioxygenase mechanism (31).
VP14 (Vi ipa ous 14), a maize enzyme media ing he oxida-
i e clea age o 9-cis- iolaxan hin and 9⬘-cis-neoxan hin o
o m he p ecu so o ABA, xan hoxin, was he i s molecu-
la ly iden i ied ca o enoid oxygenase (34). Da a om se-
quenced genomes ha e hen e ealed he occu ence o VP14
homologs in all axa, as well as o hologs in se e al plan
species. Fo ins ance, he genome o A. haliana encodes nine
membe s o his oxygenase amily (39). The A. haliana ca o -
enoid oxygenases a e in ol ed in ABA biosyn hesis (18), he
o ma ion o a s ill uniden i ied media o o he apical domi-
nance (10, 32), he syn hesis o ola ile compounds, e.g., ␤-io-
none (33), and in plas id de elopmen (25). Thus, he di e si y
o biological unc ions is mi o ed in he a ie y o he sub-
s a es, he clea age si es, and he apoca o enoids o med. In
addi ion o he A. haliana enzymes, he iden i ica ion o VP14
has also led o he cloning o u he ca o enoid oxygenases
in ol ed in he o ma ion o apoca o enoid pigmen s, such as
bixin in Bixa o ellana (12) and sa on in C ocus sa i us (13),
and he syn hesis o ola ile compounds om se e al plan
species (36, 37).
In animals, e inal and i s de i a i es ep esen he bes
known apoca o enoids. This C
20
compound is syn hesized
h ough cen al clea age o ␤-ca o ene, a eac ion media ed by
he enzyme ␤-ca o ene oxygenase I (BCO I). The co espond-
ing cDNAs we e cloned om se e al species, i.e., D osophila
melanogas e (42), chickens (44), and mammals (27, 46), allow-
ing he cha ac e iza ion o his enzyme. In addi ion o BCO I,
mammals con ain wo di e en membe s o he ca o enoid
oxygenase amily. The i s one, BCO II, media es he o ma-
ion o he ola ile compound ␤-ionone om ␤-ca o ene
h ough asymme ical clea age a he posi ion 9⬘,10⬘(19),
while he second membe , RPE65 ( e inal pigmen epi helium
65), ac s as a chape one o e inol and e inyles e a he han
as a clea age enzyme (45).
Ca o enoid-p oducing mic oo ganisms ha e been ex en-
si ely used as model sys ems in he in es iga ion o he bio-
chemis y and gene ics o his biosyn he ic pa hway ( e iewed
in e e ence 35). Se e al examples a e ep esen ed by ilamen-
* Co esponding au ho . Mailing add ess: Facul y o Biology, Albe -
Ludwigs Uni e si y o F eibu g, Schaenzles . 1, D-79104 F eibu g, Ge -
many. Phone: 49 761 203 8454. Fax: 49 761 203 2675. E-mail: salim.albabili
@biologie.uni- eibu g.de.
䌤
Published ahead o p in on 9 Feb ua y 2007.
650
on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://ec.asm.o g/Downloaded om
ous ungi, like he zygomyce es Phycomyces blakesleeanus and
Muco ci cinelloides o ␤-ca o ene o ma ion, o he ascomy-
ce es Neu ospo a c assa and Fusa ium ujiku oi o neu ospo-
axan hin biosyn hesis ( e iewed in e e ences 4 and 35). The
syn hesis o neu ospo axan hin, a C
35
acidic apoca o enoid, is
achie ed om ge anyl-ge anyl-diphospha e by i e enzyma ic
ac i i ies: he bi unc ional enzyme phy oene syn hase/lycopene
cyclase (Ca RA), he phy oene desa u ase Ca B (23), he
o ulene clea ing oxygenase (Ca T), and a p esumed dehyd o-
genase. The i s wo enzymes media e he eac ions needed o
p oduce o ulene: he condensa ion o wo ge anyl-ge anyl-
diphospha e molecules, i e desa u a ions, and he in oduc ion
o one ␤-ionone ing. The nex s ep is media ed by Ca T, which
clea es o ulene o ␤-apo-4⬘-ca o enal (26a). Finally, he
aldehyde is oxidized o he acid neu ospo axan hin by an un-
known enzyme. In addi ion o neu ospo axan hin, F. ujiku oi
accumula es mino amoun s o o ulene and ␥- and ␤-ca o ene
(2), wi h he la e being a inal side-p oduc o he Ca RA-ca a-
lyzed cycliza ion o ␥-ca o ene.
As deduced om he p esence o opsins, he ca o enoid
pa hway o he ilamen ous ungi is also supposed o be he
sou ce o an addi ional apoca o enoid, e inal. Opsins a e a
class o se en- ansmemb ane helices p o eins ha bind his
apoca o enoid ia a conse ed lysine esidue. The ligh -medi-
a ed isome iza ion o he ch omopho e enables opsins o unc-
ion as ion pumps o ligh senso y ecep o s in animals, a -
chaea, algae ( e iewed in e e ences 16 and 38), and as
ecen ly shown, in eubac e ia (6, 14), including cyanobac e ia
(41). In addi ion, opsin-encoding sequences ha e been iden i-
ied in he genomes o he ungi Neu ospo a c assa (8), Fusa -
ium ujiku oi (26), and Lep osphae ia maculans (17), bu mo e
examples a e appea ing as new ungal genomes become a ail-
able. Recen ly, i has been demons a ed ha he Lep ospha-
e ia opsin ac s as a bac e io hodopsin-like p o on pump (43). I
was also shown ha he Neu ospo a opsin NOP-1, when he -
e ologously exp essed in he yeas Pichia pas o is, o ms an
ac i e pho o ecep o exhibi ing simila cha ac e is ics o he
a chaeal senso y hodopsin II (7, 9). Howe e , he dis up ion
o nop-1did no lead o de ec able changes in pho o egula ed
p ocesses, like conida ion o pigmen a ion (8). The e o e, he
a ge p ocesses egula ed by NOP-1 a e s ill unknown.
In e es ingly, he genes esponsible o he i s s eps in he
ca o enoid biosyn hesis (ca RA and ca B) cons i u e a gene
clus e wi h wo u he membe s, ca O and ca X, coding o an
opsin-like p o ein and a pu a i e ca o enoid oxygenase, espec-
i ely. The ou genes exhibi he same ansc ip ional pa e n,
being exp essed a low le els in he da k and induced by illu-
mina ion o each a maximum a e 1ho exposu e. In addi-
ion, all ou genes a e de egula ed in ca o enoid-o e p oduc-
ing mu an s (23, 26, 40).
As men ioned abo e, ca o enoid biosyn hesis in Fusa ium
ujiku oi is induced by ligh (3). The occu ence o a gene
encoding he opsin-like p o ein Ca O in he ca gene clus e
sugges ed a possible ole o his pho o ecep o in he ligh
egula ion o he pa hway. Howe e , like in he case o NOP-1
om Neu ospo a (8), a ge ed dele ion o ca O did no lead o
a de ec able pheno ype (26). In con as o ca O, he dis up-
ion o ca X, which encodes a pu a i e ca o enoid oxygenase,
esul ed in a signi ican induc ion o ca o enoid biosyn hesis
(40), indica ing ha he enzyme is in ol ed in nega i e eed-
back egula ion o he pa hway and no in neu ospo axan hin
o ma ion, as p esumed be o e. The ca gene clus e esembles
he ecen ly desc ibed clus e s om ␥-p o eobac e ia in ha i
ha bo s genes esponsible o ␤-ca o ene o ma ion and wo
genes encoding an opsin and a e inal- o ming enzyme (29).
In he ligh o hese indings, we in es iga ed he abili y o
Ca X o clea e ca o enes. He e we epo on Ca X as he i s
e inal- o ming enzyme om ungi as deduced om in i o
and in i o s udies.
MATERIALS AND METHODS
Cloning o ca X.Fi e mic og ams o o al RNA, isola ed om mycelia g own
o 1 h unde whi e ligh , was used o cDNA syn hesis using Supe Sc ip
RnaseH
⫺
e e se ansc ip ase (In i ogen, Paisley, Uni ed Kingdom) acco ding
o he ins uc ions o he manu ac u e . Two mic oli e s o cDNA was hen
applied o he ampli ica ion o ca X using he ollowing p ime s: Ca X-1, 5⬘-A
TGAAGTTTCTGCAACAAAATTCC-3⬘; Ca X-2, 5⬘-TCATCCAACAGCTTT
CTCCAACTTC-3⬘. The PCR was pe o med using 100 ng o each p ime , 200
␮M concen a ions o deoxynucleoside iphospha es, and 1 ␮l Ad an age cDNA
polyme ase mix (BD Biosciences) in he bu e p o ided, as ollows: 2 min o
ini ial dena u a ion a 94°C, 32 cycles (30 s a 94°C, 30 s a 54°C, 2 min a 68°C),
and 10 min o inal polyme iza ion a 68°C. The ob ained PCR p oduc was
pu i ied using GFX PCR DNA and Gel Band Pu i ica ion ki (Ame sham Bio-
sciences, NJ) and cloned in o he pCR2.1-TOPO and pBAD/TOPO (In i ogen,
Paisley, Uni ed Kingdom) ec o s o yield pCR-Ca X and pBAD-Ca X, espec-
i ely. The na u e o he p oduc was e i ied by sequencing.
In i o es using ␤-ca o ene-accumula ing Esche ichia coli cells. ␤-Ca o ene-
and lycopene-accumula ing E. coli XL1-Blue cells we e gene a ed by in oduc-
ion o he plasmids pBe a and pLyc, de i a i es o pACYC177 ha bo ing he
equi ed biosyn he ic genes (c E,c B, and c I in pLyc; c E,c B,c I, and c Y
in pBe a) om E winia he bicola, allowing he o ma ion o lycopene and ␤-ca -
o ene, espec i ely. Cells we e hen ans o med wi h pBAD-Ca X. O e nigh
cul u es o he ob ained s ain ␤-Ca X and he co esponding con ol ␤-Con,
con aining Ca X in he an isense o ien a ion, we e inocula ed in o LB medium,
g own a 28°C o an op ical densi y a 600 nm o 0.5, and induced wi h 0.08%
a abinose. Cells we e hen ha es ed a e 4 h. Ca o enoids and e inoids
we e ex ac ed using o maldehyde acco ding o he me hod o on Lin ig and
Vog (42).
P o ein exp ession and pu i ica ion. To exp ess Ca X as a glu a hione S-
ans e ase (GST) usion, he co esponding cDNA was excised as a blun -end
agmen om pCR-Ca X and hen liga ed in o SmaI-diges ed and alkaline
phospha ase- ea ed pGEX-4T-1 (Ame sham Biosciences, NJ) o yield pGEX-
Ca X. The blun -end Ca X agmen was ob ained h ough No I diges ion,
ollowed by a T4 DNA polyme ase ea men and Ecl136II diges ion. Subse-
quen ly, E. coli BL21 cells we e ans o med wi h pGEX-Ca X, g own a 28°C in
2⫻YT medium, and induced a an op ical densi y a 600 nm o 0.5 wi h 0.2 mM
isop opyl-␤-D- hiogalac opy anoside (IPTG). A e o e nigh incuba ion a
18°C, cells we e ha es ed by cen i uga ion. The usion p o ein was hen pu i ied
using glu a hione-Sepha ose 4B (Ame sham Biosciences, NJ) acco ding o he
ins uc ions o he manu ac u e . The usion p o ein was elu ed wice wi h
elu ion bu e (50 mM T is-HCl, pH 8.0, 100 mM NaCl, 10 mM glu a hione,
0.2% T i on X-100 [ ol/ ol]) o 20 min a oom empe a u e. Pu i ica ion s eps
and p o ein exp ession we e analyzed by sodium dodecyl sul a e-polyac ylamide
gel elec opho esis. The con ol s ain ca ied an an isense plasmid exp essing
only GST.
P epa a ion o he subs a es. ␤-Ca o ene and lycopene we e ob ained om
Sigma-Ald ich (Deisenho en, Ge many) and Ro h (Ka ls uhe, Ge many).
␤-Apo-8⬘-ca o enal and ␤-apo-4⬘-ca o enal we e kindly p o ided by BASF, Lud-
wigsha en, Ge many. The subs a es we e pu i ied using hin-laye ch oma og-
aphy (TLC) silica gel pla es (Me ck, Da ms ad , Ge many), de eloped in pe-
oleum benzene-die hyle he -ace one (40:10:10, ol/ ol/ ol). Highly pu e
␥-ca o ene was ob ained om Ca o ena u e (Lupsingen, Swi ze land). To ulene
and neu ospo axan hin we e pu i ied om ca o enoid ex ac s o Fusa ium u-
jiku oi s ains accumula ing hese subs a es by p epa a i e high-pe o mance
liquid ch oma og aphy (HPLC), as desc ibed below.
Enzyme assays. To p oduce micelles, subs a es we e d ied using a acuum
cen i uge, esuspended in 200 ␮l pe oleum benzene, and hen mixed wi h 150
␮l o e hanolic de e gen mix u e consis ing o 0.7% ( ol/ ol) T i on X-100 and
1.6% ( ol/ ol) T i on X-405. The mix u e was d ied again and esuspended in
110 ␮l o incuba ion bu e consis ing o 200 mM HEPES-NaOH, pH 8.0, 2 mM
VOL. 6, 2007 RETINAL FORMATION IN FUNGI 651
on Feb ua y 23, 2016 by USE/BCTA.GEN UNIVERSITARIAh p://ec.asm.o g/Downloaded om
TCEP [T is(2-ca boxy-e hyl)phosphine hyd ochlo ide; ob ained om Sigma-Al-
d ich (Deisenho en, Ge many)], 0.4 mM FeSO
4
,and2mgml
⫺1
ca alase (Sigma-
Ald ich, Deisenho en, Ge many). One hund ed mic oli e s o he p epa ed mi-
celles was hen used o an in i o assay in a o al olume o 200 ␮l con aining
Ca X and he ca o ene subs a e in inal concen a ions o 200 ng ␮l
⫺1
and 20
␮M, espec i ely. A e an incuba ion o 2ha 27°C, assays we e s opped by
adding 1 olume o ace one, ex ac ed wi h pe oleum benzene-die hyle he (1:4,
ol/ ol), and subjec ed o HPLC analyses.
Pho ome ic measu emen s. Subs a es we e quan i ied spec opho ome i-
cally a hei indi idual ␭
max
using ex inc ion coe icien s calcula ed om E1% (5,
13a). P o ein concen a ion was de e mined using he Bio-Rad p o ein assay ki
(Bio-Rad, CA).
HPLC analyses. HPLC sepa a ions we e pe o med h ough a C
30
e e sed-
phase column (YMC Eu ope, Sche mbeck, Ge many) in a Wa e s sys em
(Eschbo n, Ge many) equipped wi h a pho odiode a ay de ec o (model 996).
Clea age p oduc s we e analyzed using he sol en sys ems B, me hanol
(MeOH)– e -bu ylme hyl e he –wa e (600:120:120, ol/ ol/ ol), and A, MeOH–
e -bu ylme hyl e he (500:500, ol/ ol). The column was de eloped a a low
a e o 1 ml min
⫺1
, wi h a linea g adien om 100% B o 43% B wi hin 45 min,
hen o 0% B wi hin 1 min, and main aining he inal condi ions o ano he 26
min a a low a e o 2 ml min
⫺1
.
To ulene and neu ospo axan hin we e pu i ied om o al ca o enoid ex ac s
o Fusa ium ujiku oi using he sol en sys ems B, MeOH– e -bu ylme hyl e he –
wa e (120:4:40, ol/ ol/ ol), and A, MeOH– e -bu ylme hyl e he (500:500, ol/
ol). The column was de eloped a a low a e o 1 ml min
⫺1
, wi h a linea
g adien om 100% B o 50% B wi hin 5 min, hen o 0% B wi hin 1 min, and
main aining he inal condi ions o ano he 26 min a a low a e o 2 ml min
⫺1
.
GC-MS analyses. The p oduc s ob ained om ␤-Ca X cells we e p epu i ied by
TLC using chlo o o m-washed RP-18 F
245s
pla es (Me ck, Da ms ad , Ge many).
The pla es we e de eloped in MeOH-wa e (100:1, ol/ ol), and p oduc s we e hen
sc aped o and elu ed wi h CHCl
3
, e apo a ed, and edissol ed in ace one. Gas
ch oma og aphy (GC)-mass spec ome y (MS) analyses we e pe o med using a
Finnigan T ace DSQ mass spec ome e coupled o a T ace GC gas ch oma og aph.
Sepa a ions we e ca ied ou using a 30-m Zeb on ZB5 column (5% phenyl–95%
dime hylpolysilanoxane, 0.25-mm inne diame e , and 0.25-␮m ilm hickness; Phe-
nomenex, Ascha enbu g, Ge many). Fo he iden i ica ion o e inal, a empe a u e
p og am was applied wi h an ini ial empe a u e o 100°C o 5 min, ollowed by a
empe a u e amp o 25°C min
⫺1
o a inal empe a u e o 320°C, which was main-
ained o an addi ional 5 min. A cons an He ca ie gas low was main ained a 1
ml min
⫺1
using a spli low o 1:30. The spli less ime was 3 min, and he injec o
o en empe a u e was 220°C. S anda d elec on impac ioniza ion was used a an ion
sou ce po en ial o 70 eV a 200°C. Iden i ica ion o e inal was done by ch oma o-
g aphic compa ison wi h he au hen ic e e ences and by compa ing he mass spec a
wi h he NIST Mass Spec al Sea ch P og am e sion 2.0 (Na ional Ins i u e o
S anda ds and Technology).
RESULTS
Ca X con e s ␤-ca o ene in o e inal in i o. Sequence
compa isons sugges ed ha Ca X is a membe o he ca o -
enoid oxygenase amily (40). To in es iga e i s abili y o clea e
ca o enoids, we exp essed Ca X in lycopene- and ␤-ca o ene-
accumula ing E. coli cells using he pBAD/TOPO exp ession
sys em (In i ogen, Paisley, Uni ed Kingdom). While no enzy-
ma ic ac i i y was de ec able in he lycopene backg ound, he
exp ession o Ca X in ␤-ca o ene-accumula ing cells un eiled
i asa␤-ca o ene-clea ing enzyme. As shown in Fig. 1, he
exp ession o Ca X led o an ob ious decolo iza ion o he
accumula ing cells, indica ing ␤-ca o ene clea age. The subse-
quen HPLC analyses (Fig. 1) showed ha Ca X clea ed ␤-ca -
o ene in o a compound esembling e inal in i s UV- isible
ligh spec um and elu ion p o ile. In addi ion o he aldehyde,
he ex ac con ained wo compounds likely o be he co e-
sponding alcohol e inol and i s co esponding es e . To p o e
he iden i y o he o med aldehyde, cell ex ac s we e sepa-
a ed using TLC, and he co esponding p oduc was pu i ied
and subjec ed o GC-MS analyses using e inal as a s anda d.
The p oduc o Ca X and he e inal s anda d showed iden ical
e en ion imes and, as shown in Fig. 2, he mass spec um was
iden ical o published da a (28) and o spec a in he NIST
da abase, including he p esence o he co ec molecula ion
o m/z⫽284 as well as he ypical m/z⫽161 agmen , indic-
a i e o he loss o he ionone ing om he pa en ion. These
da a demons a e ha Ca X con e s ␤-ca o ene in o e inal in
i o.
Ca X syn hesizes e inal om ␤-ca o ene, ␥-ca o ene, and
o ulene in i o. To in es iga e he enzyme ac i i y in i o,
Ca X was exp essed and pu i ied as a GST usion p o ein (Fig.
3). The pu i ied enzyme was hen used o in i o assays wi h
␤-ca o ene as a subs a e. The HPLC analysis o he in i o
assays (Fig. 4) shows a clea con e sion o ␤-ca o ene in o
e inal.
The exp ession o Ca X in lycopene-accumula ing E. coli
cells did no lead o any de ec able clea age ac i i y, indica ing
he need o a ␤-ionone ing in he ca o enoid subs a e. To
de e mine whe he monocyclic ca o enes a e also clea ed by
Ca X, in i o assays we e pe o med wi h ␥-ca o ene and
o ulene (Fig. 4). As shown in he HPLC analysis (Fig. 4),
Ca X clea ed ␥-ca o ene and o ulene in o e inal and he
co esponding acyclic aldehydes acyclo e inal and 3,4-didehy-
d o-acyclo e inal, espe i ely. The s uc u es o applied sub-
s a es and ob ained p oduc s a e gi en in Fig. 5.
Ca X con e s he syn he ic compound ␤-apo-8ⴕ-ca o enal
bu no C
35
-apoca o enoids. Recen ly, we epo ed ha he
cyanobac e ial enzymes Synechocys is and Nos oc apoca o -
enoid oxygenase con e apoca o enoids in o e inal and e i-
nal-like compounds by clea ing subs a es wi h di e en chain
leng hs a he cen al 15-15⬘double bond (28, 30). To in es i-
ga e he abili y o Ca X o p oduce e inal om apoca o enals,
we pe o med in i o assays using ␤-apo-8⬘-ca o enal (C
30
)as
a subs a e. As shown in he HPLC analysis (Fig. 4), he incu-
ba ion wi h he pu i ied Ca X enzyme esul ed in he o ma-
ion o wo p oduc s which ma ch hose o med by he Syn-
echocys is apoca o enoid oxygenase in hei UV- isible ligh
spec um and ch oma og aphic beha io (28). Thus, Ca X
con e ed he C
30
subs a e in o e inal (C
20
) and he co e-
sponding C
10
compound apo-8⬘,15⬘-apo-ca o ene-dial (2,6-
FIG. 1. In i o es o Ca X ac i i y. Ca X was exp essed in ␤-ca -
o ene-accumula ing E. coli cells (␤-Ca X) unde he con ol o an
a abinose-inducible p omo e . The induc ion led o clea decolo iza-
ion o ␤-Ca X compa ed o he con ol cells (␤-Con). The HPLC
analysis e ealed he o ma ion o p oduc (A) showing a e inal UV-
isible ligh spec um and elu ion beha io . In addi ion, a iable
amoun s o e inyl es e (B) and e inol (no shown), coelu ing wi h
e inal in he used HPLC sys em, we e p oduced.
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dime hyl-oc a-2,4,6- ien-dial, C
10
O
2
H
11
). The s uc u es o
he subs a e and he ob ained p oduc s a e gi en in Fig. 5.
Based on he con e sion o ␤-apo-8⬘-ca o enal in o e inal,
we assumed ha Ca X may also clea e he acidic apoca o -
enoid neu ospo axan hin (C
35
), he majo ca o enoid in Fusa -
ium, as well as i s co esponding aldehyde ␤-apo-4⬘-ca o enal.
The e o e, we pe o med in i o assays using hese apoca o -
enoids as subs a es. Howe e , subsequen HPLC analyses e-
ealed, in bo h cases, he o ma ion o only aces o e inal
(da a no shown).
DISCUSSION
In his wo k, we show in i o and in i o ha he ungal
enzyme Ca X is a ca o enoid oxygenase which ca alyzes he
o ma ion o e inal h ough cen al clea age o ␤-ca o ene a
FIG. 3. Coomassie-s ained sodium dodecyl sul a e gel showing
GST-Ca X pu i ica ion. Lanes: 1, o al lysa e o con ol cells exp ess-
ing GST (con ol); 2, o al lysa e o cells exp essing GST-Ca X; 3,
co esponds o sample 2 a e emo al o inclusion bodies; 4, nonbind-
ing supe na an o sample 3; 5, 4.5-␮g p o ein o he elu ion ac ion o
he con ol; 6, 4.5-␮g p o ein o elu ed GST-Ca X.
FIG. 2. GC-MS analysis o e inal p oduced in ␤-Ca X cells. (A) The elec on impac mass spec um o compound (A) (Fig. 1) showed iden i y
wi h he e e ence spec um o e inal (B) and exhibi ed he expec ed molecula ion o m/z⫽284 as well as he ypical m/z⫽161 agmen ,
indica i e o loss o he ␤-ionone ing om he pa en ion.
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he 15-15⬘double bond. To de e mine whe he monocyclic
ca o enes a e also clea ed by Ca X, in i o assays we e pe -
o med wi h ␥-ca o ene and o ulene (Fig. 4), wo compounds
also ound in F. ujiku oi (35). In a con ol expe imen , we also
pe o med an in i o assay wi h lycopene as a subs a e. The
subsequen analyses p o ed ha lycopene does no ep esen a
sui able subs a e (da a no shown). Simila esul s we e e-
po ed o he human BCO I, which clea ed ␤-ca o ene bu no
lycopene (22). In con as , he ecombinan enzyme ca alyzed
he clea age o ␥-ca o ene and o ulene in o e inal and he
co esponding acyclic aldehydes acyclo e inal and 3,4-didehy-
d o-acyclo e inal. These da a sugges ha he occu ence o a
leas one ␤-ionone ing in he subs a e is equi ed o he
clea age eac ion.
Ca o enoids may be a ge s o des uc ion p ocesses caused
by oxida i e s ess and leading o he o ma ion o apoca o -
enoids wi h di e en chain leng hs. These unspeci ic clea age
p oduc s may ep esen subs a es o ca o enoid oxygenases
which con e hem in o sho e apoca o enoids wi h a de ined
chain leng h. Fo ins ance, he cyanobac e ial enzymes Syn-
echocys is (28) and Nos oc apoca o enoid oxygenase (30)
clea ed se e al apoca o enoids o e inal and e inal-like com-
pounds. I was also shown ha he A. haliana enzyme
A CCD1, which clea es all- ans-ca o enoids a he 9-10 and
9⬘-10⬘double bonds, also eadily con e s ␤-apo-8⬘-ca o enal
(C
30
) in o ␤-ionone (C
30
)andaC
17
-dialdehyde (31). In ligh o
hese indings, we in es iga ed he abili y o Ca X o clea e
␤-apo-8⬘-ca o enal and obse ed a clea con e sion o his
subs a e in o e inal and apo-8⬘,15⬘-apo-ca o ene-dial. This
sugges s ha he 15-15⬘double bond is he a ge o he Ca X-
media ed clea age, i espec i e o he leng h o he subs a e.
Addi ionally, he o ma ion o e inal om ␤-apo-8⬘-ca o enal
indica es ha he dis ance o he ␤-ionone ing de e mines he
clea age si e, as was shown o he apoca o enoid clea age
enzymes Synechocys is (28) and Nos oc apoca o enoid oxygen-
ase (30).
The clea age o ␤-apo-8⬘-ca o enal indica ed ha he en-
zyme may also con e he na u al C
35
apoca o enoid neu os-
po axan hin, accumula ed in Fusa ium, and i s p ecu so
␤-apo-4⬘-ca o enal. Su p isingly, in i o assays wi h hese sub-
s a es esul ed in he o ma ion o only aces o e inal. This
ob ious con adic ion o he con e sion o he C
30
compound
␤-apo-8⬘-ca o enal could be explained by in e ac ions be ween
he oxygene unc ional g oups and he subs a e-binding ca i y.
Such in e ac ions may shi he 15-15⬘clea age si e om he
eac ion cen e o impede he isome iza ion s ep, which is
assumed o occu p io clea age (20). The con e sion o he
sho e ␤-apo-8⬘-ca o enal indica es ha he occu ence o
such in e ac ions depends on he leng h o he subs a e. Elu-
cida ion o he s uc u e o Ca X may p o ide an explana ion
o he di e en ac i i ies obse ed.
The disco e y o ca X as a ou h cons i uen o he ca
clus e , which encodes wo ca o enoid biosyn hesis enzymes
(Ca RA and Ca B) and an opsin-like p o ein (Ca O), had led
o he sugges ion ha Ca X migh be esponsible o he o -
ma ion o he end p oduc , neu ospo axan hin (40), which is
syn hesized h ough he oxida i e clea age o o ulene. How-
e e , a ge ed dele ion o ca X did no esul in a loss o he
capabili y o syn hesizing neu ospo axan hin. On he con a y,
he dis up ion o ca X was accompanied by a duplica ion o he
o al ca o enoid con en unde illumina ion and by an abou
10- old inc ease in he da k (40). This signi ican enhancemen
o he ca o enoid amoun sugges s ha he p oduc o med by
Ca X is a signaling compound down egula ing ca o enoid bio-
syn hesis. Acco dingly, i was shown ha he ansc ip le els o
ca RA and ca O a e inc eased in he ca X mu an (40).
The e inal- o ming ac i i y o Ca X p esen ed he e p o-
ides a possible explana ion o he e ec s obse ed in he
⌬ca X mu an in he ligh . I can be specula ed ha Ca X
deli e s he ch omopho e e inal o an opsin, which exe s a
nega i e eedback on ca o enoid biosyn hesis ia an unknown
signaling cascade. This hypo hesis does no con adic he p e-
ious inding ha loss o unc ion o he pu a i e opsin gene
ca O om F. ujiku oi did no ha e a de ec able impac on
ca o enoid biosyn hesis (26). The absence o a ca o enogenic
pheno ype in he ca O
⫺
mu an could be explained by edun-
dancy o opsins, since he genome o F. ujiku oi encodes a
leas wo addi ional opsin-like p o eins (A. F. Es ada, pe -
FIG. 4. HPLC analyses o he in i o assay p oduc s ob ained om
␤-apo-8⬘-ca o enal (I), ␤-ca o ene (II), ␥-ca o ene (III), and o ulene
(IV) and he co esponding UV- isible ligh spec a (A o D). Ca X
o med e inal (A) om he ou subs a es (S). The con e sion o
e inal led also o he o ma ion o apo-8⬘,15⬘-apo-ca o ene-dial
(B) om ␤-apo-8⬘-ca o enal, acyclo e inal (C) om ␥-ca o ene, and
3,4-didehyd o-acyclo e inal (D) om o ulene. The s uc u e o sub-
s a es and p oduc s is gi en in Fig. 5.
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sonal communica ion). This p esump ion is suppo ed by he
occu ence o h ee opsin-like p o eins in he closely ela ed
Fusa ium g aminea um (Gibbe ella zae PH-1). Based on se-
quence homology, wo o hem (accession no. XP_383240 and
XP_387730) can be conside ed closely homologous o Ca O
(accession no. CAD97459) and he opsin-like p o ein OpsA
om F. ujiku oi (A. F. Es ada, pe sonal communica ion),
espec i ely, while a coun e pa o he hi d (accession no.
XP_381616) has no been iden i ied so a . In e es ingly, OpsA
(accession no. XP_387730) is he closes homolog o NOP-1
om Neu ospo a (8), showing a simila i y o 74% and iden i y
o 54% o e a s e ch o 278 amino acids. The exp ession o
nop1 in he yeas Pichia pas o is led o an ac i e, e inal-binding
opsin wi h abso p ion p ope ies and a pho ochemical eac ion
cycle cha ac e is ic o he a chaeal senso y hodopsins (9).
Howe e , he ⌬nop-1mu an did no show any pheno ype wi h
espec o ligh - egula ed p ocesses (8). Taken oge he , he
up- egula ion o ca o enoid biosyn hesis mani es ed by he
⌬ca X mu an in he ligh could be due o he loss o unc ion
o ano he opsin because o he lack o e inal o migh be, like
he induc ion in he da k, opsin independen . In he la e case,
i could be assumed ha he Ca X-media ed clea age eac ions
sense he ca o enoid con en and hus implies ha e inal o i s
de i a i es a e media o s o a nega i e eedback egula ion o
FIG. 5. S uc u e o subs a es and p oduc s o he in i o assays. The enzyme con e ed ␤-apo-8⬘-ca o enal (I) in o e inal (A) and
apo-8⬘,15⬘-apo-ca o ene-dial (B), ␤-ca o ene (II) in o e inal (A), ␥-ca o ene (III) in o e inal (A) and acyclo e inal (C), and o ulene (IV) in o
e inal (A) and 3,4-didehyd o-acyclo e inal (D). As indica ed, all subs a es we e clea ed a he 15-15⬘double bond.
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he pa hway. Thewes e al. (40) epo ed ha he supplemen-
a ion o ⌬ca X mu an wi h e inyl es e o e inal did no lead
o escue o he obse ed pheno ype. Howe e , ou knowledge
abou he abso p ion and me abolism o hese compounds in
Fusa ium is s ill limi ed. The e o e, he escue expe imen s
should be op imized, p obably by using adiolabeled e inal. In
addi ion, he e ec o a hi d concei able e inoid signaling
molecule, e inoic acid, should be also in es iga ed.
The p e iously obse ed ⌬ca X pheno ype and he da a p e-
sen ed he e indica e ha he e inal- o ming ac i i y is solely
ca alyzed by Ca X in F. ujiku oi. This conclusion coincides
wi h he occu ence o only wo ca o enoid oxygenases in he
closely ela ed ungus Fusa ium g aminea um. The i s (acces-
sion no. XP_383243) shows a simila i y o abou 84% o Ca X,
indica ing i s in ol emen in e inal o ma ion; he second (ac-
cession no. XP_382801) is Ca T (26a), he enzyme which ca -
alyzes he ini ial clea age o o ulene which leads o he o -
ma ion o neu ospo axan hin. No su p isingly, BLAST
analysis o he Neu ospo a genome e eals also wo ca o enoid
oxygenases in his ungus (accession no. XP_961764.1 and
XP_958452). The simila i y o he ca o enoid pa hway o Neu-
ospo a wi h ha o F. ujiku oi and he occu ence o opsins
sugges equi alen oles o he Neu ospo a ca o enoid oxyge-
nases as Ca X and Ca T coun e pa s. The ac i i ies o he
Neu ospo a enzymes a e cu en ly unde in es iga ion. The
dis up ion o ca X o hologues in o he ungal species con ain-
ing mo e han one opsin gene p o ides a powe ul ool o
un eil he unc ions o hese ligh ecep o s.
Ou esul s ep esen he i s epo on e inal biosyn hesis
in he ungal kingdom. This ou s anding enzyme ac i i y,
needed o he p oduc ion o i amin A and e inoic acid in
animals, was o me ly ound in e y dis an species, om eu-
bac e ia and a chaea o highe euka yo es. The occu ence o
a e inal-p oducing enzyme also in a ilamen ous ungus p o-
ides aluable e idence on hei ubiqui y in all he majo ax-
onomic g oups and on hei sequence conse a ion and opens
he way o hei iden i ica ion in o he ungi and lowe eu-
ka yo es.
ACKNOWLEDGMENTS
We hank Pe e Beye o aluable discussions, Jo ge Maye o
co ec ing he English e sion o he manusc ip , and Hansgeo g E ns
o p o iding he syn he ic apoca o enoids.
This wo k was suppo ed by a g an o Pe e Beye by he Bill &
Melinda Ga es Founda ion as pa o he G and Challenges in Global
Heal h Ini ia i e and Deu sche Fo schungsgemeinscha (DFG) g an
AL892-1. A.P.-C. was suppo ed by a sho - e m EMBO ellowship.
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