JOURNAL OF BACTERIOLOGY,
0021-9193/97/$04.0010July 1997, p. 4513–4522 Vol. 179, No. 14
Copy igh © 1997, Ame ican Socie y o Mic obiology
Func ional Complemen a ion o an Esche ichia coli gap Mu an
Suppo s an Amphibolic Role o NAD(P)-Dependen
Glyce aldehyde-3-Phospha e Dehyd ogenase o
Synechocys is sp. S ain PCC 6803
FEDERICO VALVERDE, MANUEL LOSADA, AND AURELIO SERRANO*
Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Cen o de In es igacio´n Isla de la Ca uja,
Uni e sidad de Se illa-CSIC, E-41092 Se ille, Spain
Recei ed 7 Janua y 1997/Accep ed 30 Ap il 1997
The gap-2 gene, encoding he NAD(P)-dependen D-glyce aldehyde-3-phospha e dehyd ogenase (GAPDH2)
o he cyanobac e ium Synechocys is sp. s ain PCC 6803, was cloned by unc ional complemen a ion o an
Esche ichia coli gap mu an wi h a genomic DNA lib a y; his is he i s ime ha his cloning s a egy has been
used o a GAPDH in ol ed in pho osyn he ic ca bon assimila ion. The Synechocys is DNA egion able o
complemen he E. coli gap mu an was na owed down o 3 kb and ully sequenced. A single comple e open
eading ame o 1,011 bp encoding a p o ein o 337 amino acids was ound and iden i ied as he pu a i e gap-2
gene iden i ied in he comple e genome sequence o his o ganism. De e mina ion o he ansc ip ional s a
poin , iden i ica ion o pu a i e p omo e and e mina o si es, and o ien a ion o he unca ed lanking genes
sugges ed he gap-2 ansc ip should be monocys onic, a possibili y u he con i med by No he n blo
s udies. Bo h na u al and ecombinan homo e ame ic GAPDH2s we e pu i ied and ound o exhibi i ually
iden ical physicochemical and kine ic p ope ies. The ecombinan GAPDH2 showed he dual py idine nucle-
o ide speci ici y cha ac e is ic o he na i e cyanobac e ial enzyme, and simila a ios o NAD- o NADP-
dependen ac i i ies we e ound in cell ex ac s om Synechocys is as well as in hose om he complemen ed
E. coli clones. The deduced amino acid sequence o Synechocys is GAPDH2 p esen ed a high deg ee o iden i y
wi h sequences o he chlo oplas ic NADP-dependen enzymes. In ag eemen wi h his esul , immunoblo
analysis using monospeci ic an ibodies aised agains GAPDH2 showed he p esence o he 38-kDa GAPDH
subuni no only in c ude ex ac s om he gap-2-exp essing E. coli clones and all cyanobac e ia ha we e
es ed bu also in hose om euka yo ic mic oalgae and plan s. Wes e n and No he n blo expe imen s showed
ha gap-2 is conspicuously exp essed, al hough a di e en le els, in Synechocys is cells g own in di e en
me abolic egimens, e en unde chemohe e o ophic condi ions. A possible amphibolic ole o he cyanobac-
e ial GAPDH2, namely, anabolic o pho osyn he ic ca bon assimila ion and ca abolic o ca bohyd a e
deg ada i e pa hways, is discussed.
Phospho yla ing D-glyce aldehyde-3-phospha e dehyd oge-
nases (GAPDHs) a e enzymes in ol ed in he cen al pa hways
o ca bon me abolism ha ha e been ound in all o ganisms
s udied so a (16). Due o hei key me abolic oles, hese
p o eins and hei co esponding gap genes ha e been well
cha ac e ized in many o ganisms (8, 16). In he case o he
widesp ead glycoly ic GAPDH, by a he bes -s udied membe
o his enzyme amily, he knowledge o i s s uc u e has al-
lowed di ec ed mu agenesis o essen ial amino acid esidues o
he ac i e si e and he e en ual de ailed desc ip ion o he
enzyma ic mechanism (12, 18, 44).
Th ee dis inc GAPDH enzymes wi h di e en subcellula
localiza ions and pe o ming di e se oles a e p esen in pho-
osyn he ic euka yo ic o ganisms: (i) a ypical NAD-dependen
glycoly ic enzyme (EC 1.2.1.12), simila o ha ound in all
o ganisms so a s udied and loca ed in he cy oplasm; (ii) he
NADP-dependen GAPDH (EC 1.2.1.13), a key componen o
he educ i e pen ose phospha e (RPP) cycle, which is loca ed
in he chlo oplas s oma (7, 42); and (iii) a cy osolic nonphos-
pho yla ing NADP-dependen GAPDH (EC 1.2.1.9), cha ac-
e is ic o pho osyn he ic euka yo es, ha has been p oposed
o me abolize ioses expo ed om he chlo oplas bu whose
p ecise unc ion emains o be es ablished (29, 41). In con as ,
a single GAPDH enzyme exhibi ing compa able ac i i y le els
wi h NAD o NADP as he co ac o (EC 1.2.1.-) has been
ound in pho oau o ophic cyanobac e ia and claimed, on he
basis o p elimina y biochemical da a, o be in ol ed in bo h
pho osyn he ic (anabolic) and glycoly ic (ca abolic) ca bon me-
abolism (23). Howe e , ecen epo s on he exis ence o
di e en gap genes in cyanobac e ia (8, 28, 48) aised he
ques ion o hei unc ionali y and possible physiological oles
o hei p oduc s. On he o he hand, he possible exis ence o
a single NAD(P)-dependen GAPDH in he uncompa -
men ed cyanobac e ial cell, whose cha ac e is ics a e ye o be
ully desc ibed, aised se e al as ye unanswe ed ques ions,
such as i s in ol emen in bo h pho osyn he ic and deg ada i e
ca bon me abolism, i s egula ion a he p o ein o RNA le el,
and i s ela ionships wi h he me abolism o o he essen ial
bioelemen s like N and P. This NAD(P)-dependen enzyme,
which we ha e called GAPDH2, could also p o ide a good
sys em wi h which o in es iga e a he molecula le el he basis
o NAD o NADP speci ici y. Fu he mo e, since he gap genes
ha e been ex ensi ely used in e olu iona y s udies (8, 16, 28),
an ex ensi e knowledge o he cyanobac e ial gap genes migh
* Co esponding au ho . Mailing add ess: Ins i u o de Bioquı´mica
Vege al y Fo osı´n esis, Uni e sidad de Se illa-CSIC, Cen o de In es-
igacio´n Isla de la Ca uja, A . Ame ico Vespuccio s/n, E-41092
Se ille, Spain. Phone: 34-5-4489524. Fax: 34-5-4460065. E-mail: au e-
[email p o ec ed].
4513
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help o elucida e chlo oplas e olu ion and pho osyn hesis de-
elopmen .
In his wo k, he gap-2 gene encoding GAPDH2, whose
anabolic ole in pho osyn he ic ca bon assimila ion is well es-
ablished, was cloned by means o unc ional complemen a ion
o an Esche ichia coli gap mu an om Synechocys is sp. s ain
PCC 6803, a unicellula cyanobac e ium able o g ow he e o-
ophically. This cloning s a egy, p e iously used o ypical
glycoly ic gap genes (5, 11, 51), has allowed us o show ha he
cyanobac e ial GAPDH2 is able o e icien ly pe o m a ca a-
bolic ole in i o, aising he ques ion o whe he i is an
amphibolic o dual-ope a ing enzyme in Synechocys is sp.
s ain PCC 6803. Despi e he inding o wo di e en gap genes
in he Synechocys is sp. s ain PCC 6803 genome (25), nei he
hey no hei p oduc s ha e been cha ac e ized so a , and a
de ailed s udy o hei possible egula ion by ophic condi ions
emained o be done. The exp ession o gap-2 in Synechocys is
sp. s ain PCC 6803 has been analyzed a bo h he p o ein and
mRNA le els, and he GAPDH2 has been pu i ied and kine -
ically cha ac e ized. In ag eemen wi h he amphibolic cha ac-
e p oposed o his p o ein in Synechocys is sp. s ain PCC
6803, he gap-2 p oduc is conspicuously p esen unde all
cul u al condi ions es ed, e en in he e o ophically g own
cells.
MATERIALS AND METHODS
O ganisms and g ow h condi ions. Synechocys is sp. s ain PCC 6803 and he
o he cyanobac e ial s ains used in his s udy (see Table 2) we e cul u ed a 30°C
in BG11 medium (34) supplemen ed wi h1go sodium bica bona e pe li e and
bubbled wi h 1.5% ( ol/ ol) CO
2
in ai unde con inuous whi e ligh (25 W/m
2
).
When indica ed, he usual ni ogen sou ce (NaNO
3
, 20 mM) was omi ed (N-
s a ed cells) o eplaced wi h 15 mM NH
4
Cl bu e ed wi h 30 mM N- is(hy-
d oxyme hyl)me hyl-2-aminoe hane sul onic acid (TES)–NaOH (pH 7.5). When
glucose was added o he cul u es, i was supplied a a inal concen a ion o 10
mM. Cells cul u ed he e o ophically wi h glucose we e g own in da kness and
subjec ed o a 5-min whi e ligh ac i a ion pe iod pe day, a ea men needed in
his case bu one ha did no p omo e pho osyn he ic g ow h (1). Cells cul u ed
in BG11 medium wi hou sodium phospha e (s anda d concen a ion was 0.2
mM) o 4 days we e conside ed phospha e-limi ed cells (19). A e his ime,
cul u es emained iable since g ow h esumed, a e a lag, upon eaddi ion o
phospha e. All o he cyanobac e ial s ains we e g own pho oau o ophically in
no mal BG11 medium wi h ni a e as he ni ogen sou ce.
Func ional complemen a ion expe imen s we e ini ially pe o med wi h he E.
coli gap s ain DF221 (22), ob ained om he E. coli Gene ic S ock Cen e (Yale
Uni e si y), which con ains an ambe mu a ion in he gap-1 gene. Ano he E. coli
gap mu an , W3CG, in which his gene is inac i a ed by ansposon Tn10 inse -
ion was la e used (18). Bo h s ains we e cul u ed in M63 medium (22) sup-
plemen ed wi h succina e (0.4%, w / ol) and glyce ol (0.1%, ol/ ol) o , in
complemen a ion s udies, wi h glucose (0.4%, w / ol) and, when necessa y, am-
picillin (50 mg/ml) and e acycline (35 mg/ml). E. coli K-12 o DH5awas
cul u ed ei he in M63 plus glucose o in Lu ia b o h (LB) medium, supple-
men ed when necessa y wi h ampicillin (100 mg/ml). M63 plus glucose and LB,
in which he gap mu an s we e unable o g ow, we e used, supplemen ed wi h he
app op ia e an ibio ics, as selec i e media o check o unc ional complemen-
a ion. All E. coli cells we e g own a 37°C wi h con inuous shaking a 200 pm.
Enzyme assays and p o ein echniques. All eagen s we e o analy ic g ade
and we e pu chased om Sigma Chemical Co. (S . Louis, Mo.). D-Glyce alde-
hyde-3-phospha e (G3P) dehyd ogenase (phospho yla ing) ac i i y was de e -
mined spec opho ome ically a 30°C by moni o ing ei he he gene a ion ( o -
wa d di ec ion, oxida ion o G3P in he p esence o sodium a sena e) o
disappea ance ( e e se eac ion, educ ion o 1,3-bisphosphoglyce a e) o
NAD(P)H a 340 nm (29). A coupled assay, in which aldolase (1 U/ml) p o-
duced he s oichiome ic b eakage o uc ose-1,6-bisphospha e (1 mM) o G3P
and dihyd oxyace one-phospha e, he i s p oduc being he ac ual subs a e o
he oxida i e eac ion (40), was usually used du ing enzyme pu i ica ion. Fo
kine ic s udies, howe e , DL-G3P in aqueous solu ion was used a a inal 1 mM
concen a ion o he D-isome . One uni o enzyme was de ined as he amoun
which ca alyzes he gene a ion, o disappea ance, o 1 mmol o NAD(P)H pe
min. The es e ase ac i i y o GAPDH was de e mined spec opho ome ically a
30°C by moni o ing he p-ni ophenol gene a ed in he hyd olysis o p-ni ophe-
nyles e s a 400 nm (44). All kine ic pa ame e s gi en ep esen a e age alues
o a leas ou independen de e mina ions.
P o ein concen a ion was es ima ed by ei he he B ad o d (4) o he Low y
(27) echnique, using o albumin as a s anda d. Elec opho esis o p o ein ex-
ac s was done by he Laemmli me hod in 12% (w / ol) ac ylamide slab gels in
he p esence o sodium dodecyl sul a e (SDS) o unde nondena u ing condi-
ions in polyac ylamide g adien gels (7.5 o 15% [w / ol] ac ylamide/0 o 30%
[ ol/ ol] glyce ol), using a Minip o ean II appa a us (Bio-Rad, Richmond, Cali .)
and Sigma MW-SDS-701 o MW-ND-500 p o ein ma ke s, espec i ely. Nonde-
na u ing gels we e s ained o NAD- o NADP-linked GAPDH ac i i y in si u by
incuba ion o 30 min in he s anda d eac ion mix u e o he oxida i e eac ion,
wi h ei he NAD o NADP, supplemen ed wi h 20 mM phenazine me hosul on-
a e and 1 mM p-ni oblue e azolium chlo ide. The enzyme band was loca ed by
he appea ance o a deep-pu ple band o insoluble o mazan. In he absence o
aldolase o uc ose 1,6-diphospha e (and he e o e G3P) o a sena e in he
s aining sys em, no colo was de eloped. Isoelec ic ocusing (35) was done wi h
he same elec opho e ic sys em in 5% (w / ol) ac ylamide slab gels holding
ampholi e-gene a ed pH g adien s (pH ange, 3.5 o 10.0; Pha mali e 3.5-10;
Pha macia Bio ech, Uppsala, Sweden), 25 mM NaOH and 20 mM HCl as
ca hode and anode solu ions, espec i ely, and he Sigma 3.6-9.3 IEF-MIX iso-
elec ic ocusing p o ein ma ke ki . Analy ical as p o ein liquid ch oma og a-
phy (FPLC; Pha macia Bio ech) gel il a ion o pu i ied GAPDH2 p epa a ions
was pe o med by isoc a ic elu ion on a Supe ose 6HR 10/30 column (1 by 30
cm), using an au oma ed Pha macia FPLC sys em. All p o ein physicochemical
pa ame e s p esen ed a e a e age alues o h ee independen de e mina ions.
Fo amino- e minal p o ein sequencing, 50 pmol o pu e p o ein, om ei he
he ecombinan o na i e GAPDH2, was esuspended in wa e and blo ed o
poly inylidenedi luo ide il e s (Millipo e, Bed o d, Mass.). The samples we e
hen subjec ed o mic osequencing by au oma ed Edman deg ada ion on a
model 476A p o ein sequence ( e sion 2.00; Applied Biosys ems, Fos e Ci y,
Cali .), and 10 amino acids om he amino- e minal end we e sequenced.
Cloning s a egy and DNA manipula ion. Res ic ion and modi ying enzymes
we e pu chased om Pha macia Bio ech, P omega (Madison, Wis.), o Boeh -
inge Mannheim (Mannheim, Ge many).
Compe en cells o E. coli DF221 we e ans o med wi h a o al genomic DNA
lib a y om Synechocys is sp. s ain PCC 6803 cloned in he ClaI si e (inse sizes
in he ange o 6 o 10 kb) o plasmid pBluesc ip SK
1
(pBS); S a agene, La
Jolla, Cali .). The ans o ma ion mix u e was cul u ed in LB liquid medium
supplemen ed wi h 50 mg o ampicillin pe ml. I g ow h ook place in he
selec i e medium, cells we e ha es ed by cen i uga ion, and an alkaline ex ac-
ion o plasmid was done (36). The plasmid was ans e ed in o E. coli DH5a o
a oid po en ial DNA ea angemen s ha may occu in s ain DF221. Fo u -
he exp ession expe imen s, and since he a e o spon aneous e e sion o he
ambe gap mu an was a he high (ca. 10
23
), he gap E. coli mu an s ain W3CG
(18) was chosen. The gap-1 gene o Anabaena sp. s ain PCC 7120 was cloned by
using he same unc ional complemen a ion s a egy, o e exp ession o he cya-
nobac e ial NAD-speci ic GAPDH1 being ound in he ecombinan E. coli clone
(49).
Ch omosomal DNA was isola ed om cyanobac e ial cells as p e iously de-
sc ibed (6). Fo DNA-DNA hyb idiza ion expe imen s (Sou he n blo ing), he
me hod o Ausubel e al. (2) was used. Samples o cyanobac e ial genomic DNA
we e comple ely diges ed wi h di e en es ic ion enzymes, un in 0.7% (w / ol)
aga ose gels, and blo ed o nylon memb anes (Ze ap obe; Bio-Rad, Richmond,
Cali .). The il e was hen p ehyb idized in a bu e (53SSC [13SSC is 0.18 M
NaCl plus 10 mM sodium ci a e {pH 7.0}], 53Denha d ’s solu ion [13Den-
ha d ’s solu ion is 0.02% {w / ol} Ficoll, 0.02% {w / ol} poly inylpy olidone,
and 0.02% {w / ol} bo ine se um albumin]) con aining 50 mg o salmon spe m
DNA pe ml and 0.1% (w / ol) SDS. Ei he an AseI es ic ion agmen o ca.
2.1 kb om he pFV8 plasmid inse , an in e nal BglII-A aI agmen o gap-2,o
a PCR-gene a ed gap-2 agmen o ca. 0.94 kb con aining 93% o he coding
sequence, ob ained om genomic DNA by using degene a e oligonucleo ides
based on highly conse ed egions o GAPDHs (16) [sense p ime , 59-AAT(C)
GGA(CGT)TTC(T)GGA(CGT)A(C)GA(G)ATA(CT)GGA(CGT)A(C)G-39;
an isense p ime , 59-ACCATG(A)CTG(A)TTG(A)CTC(T)ACC(G)CC-39],
was used as a p obe a e adiolabelling wi h
32
P (Random-P ime ki ; Boeh -
inge Mannheim). Homologous hyb idiza ion was pe o med a 65°C ollowed by
sequen ial washing o he il e a oom empe a u e wi h 23SSC–0.1% (w / ol)
SDS o 5 min, 23SSC–0.1% (w / ol) SDS o 15 min, 0.53SSC–0.1% (w / ol)
SDS o 15 min, 0.13SSC–0.1% (w / ol) SDS o 15 min, and inally 0.13
SSC–1% (w / ol) SDS o 30 min a 42°C. In he e ologous condi ions, he
hyb idiza ion empe a u e was 55°C and he inal highly s ingen washing s ep
was omi ed. The nylon il e s we e hen exposed o ilms (Kodak X-100) a
280°C and e en ually de eloped.
Fo DNA sequencing, plasmid pFV8 was subcloned by sequen ial exonuclease
dele ions (E ase a Base ki ; P omega), and esul ing plasmids we e hen se-
quenced in bo h s ands by a modi ica ion o he dideoxy-chain e mina ion
me hod (37) wi h [
35
S]dATP and Taq polyme ase (Taq T ack ki ; P omega) as
ins uc ed by he manu ac u e .
Fo o al RNA ex ac ion, a modi ica ion o he one-s ep p ocedu e o Chom-
czynski and Sacchi (10) was used as desc ibed by he manu ac u e (RNAgen s
To al RNA Isola ion Sys em; P omega) wi h cells om Synechocys is sp. s ain
PCC 6803 g own unde di e en cul u e condi ions un il mid-exponen ial phase.
Sepa a ion o RNA on o maldehyde-aga ose gels and hyb idiza ion o nylon
memb anes (Ze a-P obe; Bio-Rad) we e ca ied ou acco ding o he manu ac-
u e ’s ins uc ions; 15 mg o o al RNA was loaded pe lane. The hyb idiza ion
and washing s eps (wi h SSP [13SSP is 0.18 M NaCl plus 10 mM NaH
2
PO
4
zH
2
O;
pH 7.4] solu ion ins ead o SSC) we e hose desc ibed o he Sou he n blo p o ocol
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using ei he he same p obes o ano he one om npB co esponding o he RNA
pa o he Synechocys is sp. s ain PCC 6803 RNase P ibozyme (50) as a loading
con ol. The il e s we e hen applied o an Ins an Image Elec onic Au o adiog-
aphy appa a us (Packa d Ins umen Co., Downe s G o e, Ill.), and he esul ing
adioac i i y pe ma k was coun ed and analyzed.
P ime ex ension analysis was done wi h 10 mg o o al RNA and a 23-
nucleo ide-long oligonucleo ide p ime om a sequence o he 59 egion o he
coding sequence, eading ups eam om he complemen a y chain (59-CGTCC
GATCCGTCCAAATCCGTT-39). A ian myeloblas osis i us e e se ansc ip-
ase (P omega) was used. Deoxynucleo ides, including [a-
32
P]dCTP (3,000 Ci/
mmol), we e added a 1 mM ( inal concen a ion) in he eac ion mix u e (30
min, 42°C), so ha he inal cDNA agmen was ho oughly adiolabelled. RNA
was hen o ally diges ed by RNase ea men (30 min, 37°C), and he esul ing
p epa a ion was applied o a Sephac yl S-400 minicolumn (Pha macia Bio ech).
A s anda d sequencing eac ion o he inse o pFV8, using as he p ime he
same oligonucleo ide, was un and elec opho esed pa allel o he adiolabelled
cDNA ex ension p oduc .
Pu i ica ion o na u al and ecombinan Synechocys is sp. s ain PCC 6803
GAPDH2. Synechocys is sp. s ain PCC 6803 cells om la e loga i hmic g ow h
phase we e ha es ed by cen i uga ion a 10,000 3gin a So all RC5C cen i-
uge a 4°C and washed wice wi h 50 mM T is-HCl (pH 7.5) bu e , and he cell
pelle was s o ed a 220°C. F ozen cells we e esuspended in 50 mM T is-HCl
(pH 7.5) bu e wi h 10% ( ol/ ol) glyce ol, 10 mM b-me cap oe hanol, 1 mM
di hio h ei ol (DTT), 0.5 mM EDTA, and 5 mM po assium phospha e (s anda d
bu e ) supplemen ed wi h he p o ease inhibi o s phenylme hylsul onyl luo ide
(1 mM), ε-aminocap oic acid (2 mM), and benzamidine (0.5 mM), a a a io o
3 ml/g ( esh weigh ) o cells. The cells we e hen dis up ed by ul asonic ea -
men in a chilling wa e ba h, using a B anson 25U Soni ie a medium s eng h
(six 30-s pulses wi h in e media e 30-s pe iods o es ). The esul ing b oken-cell
suspension was hen supplemen ed wi h MgCl
2
up o 10 mM and cen i uged a
40,000 3g o 30 min. This g eenish supe na an (conside ed he c ude ex ac )
was ea ed wi h 10 mM s ep omycin sul a e o 1ha 4°Cunde con inuous
s i ing and cen i uged as desc ibed abo e, and he esul ing supe na an was
ea ed wi h solid ammonium sul a e; he p o ein ac ion ob ained by 40 o 80%
ammonium sul a e sa u a ion was collec ed. This p o ein pelle was esuspended
in a minimal olume (ca. 10 ml) o s anda d bu e , dialyzed o e nigh agains 5
li e s o 20 mM T is-HCl bu e (pH 7.5) wi h he same addi ions as he s anda d
bu e excep ha 0.1 mM DTT was used and no po assium phospha e was added
(dialysis bu e ), and hen applied a a lux o 30 ml/h o a DEAE-cellulose DE-52
(Wha man, Maids one, England) column (3 by 12 cm) p eequilib a ed wi h
s anda d bu e . A e a ho ough wash, anion-exchange ch oma og aphy was
pe o med wi h a linea g adien o po assium phospha e (5 o 200 mM, pH 7.5;
o al olume, 300 ml) in s anda d bu e . F ac ions o 4 ml we e collec ed, and
hose which showed enzyma ic ac i i y we e pooled. Solid ammonium sul a e was
added up o 25% (w / ol) o he esul ing enzyme p epa a ion, which was applied
a low lux (12 ml/h) o a phenyl-Sepha ose HP (Pha macia Bio ech) column (1.6
by 18 cm) p eequilib a ed wi h s anda d bu e wi h 25% (w / ol) ammonium
sul a e. A e ho ough washing o he column, he hyd ophobic in e ac ion
ch oma og aphy was de eloped by a double linea g adien ( o al olume, 150
ml), wi h dec easing ammonium sul a e ( om 25 o 0%, w / ol) and inc easing
glyce ol ( om 10 o 20%, ol/ ol) in s anda d bu e . The 1.9-ml ac ions o he
single ac i i y peak we e pooled, dialyzed o 3 h agains dialysis bu e , and hen
in oduced a a lux o 3 ml/h in a Blue Sepha ose CL-4B (Pha macia Bio ech)
column (1 by 5 cm) p e iously equilib a ed wi h dialysis bu e . A e ho ough
washing o he column wi h his bu e , he pH was aised o 8.5 and he
po assium phospha e concen a ion was inc eased o 50 mM. A e a wash wi h
wo column olumes o his bu e , he enzyme ac i i y was elu ed wi h a 5-ml
pulse o 10 mM NAD o NADP in dialysis bu e , and ac ions wi h maximal
ac i i y we e collec ed and pooled. This pu i ied GAPDH p epa a ion showed
only one 38-kDa p o ein band in SDS-polyac ylamide minigels. Cell ex ac s o
o he cyanobac e ia we e ob ained by using he p ocedu e desc ibed abo e o
Synechocys is sp. s ain PCC 6803 cells.
A simila p ocedu e was used o pu i ica ion o ecombinan Synechocys is sp.
s ain PCC 6803 GAPDH2 om E. coli W3CG gap mu an s ain cells ans-
o med wi h pFV8 and cul u ed in LB medium supplemen ed wi h ampicillin
(100 mg/ml) and e acycline (35 mg/ml). La e-loga i hmic-phase cells om 2
li e s o medium we e collec ed as desc ibed abo e, and he same pu i ica ion
p ocedu e was ollowed. In his case, he anion-exchange ch oma og aphy was
essen ial in o de o sepa a e he enzyme om he g ea amoun o p o eins and
lipids p esen in he E. coli c ude ex ac s.
Column ch oma o ocusing was used as bo h an analy ical and a p epa a i e
echnique o cha ac e ize he pu i ied enzyme p epa a ions and check o he
possible p esence o iso o ms. A column bed o Polybu e exchange PBE (1 by
18 cm) and a solu ion o Polybu e 74-HCl (1:10 dilu ion, adjus ed o pH 3.5)
we e used. The ac i i y was elu ed as ins uc ed by he manu ac u e (Pha macia
Bio ech) by he con inuous pH g adien ( om 7.5 o 3.5) gene a ed by washing
he column wi h he Polybu e solu ion.
Immunological echniques. A abbi was injec ed wi h 500 mg o pu e
GAPDH2 p o ein in aqueous solu ion 1:1 wi h F eund’s coadju an . A e 21
days, a small sample o blood was collec ed, and a second dose o 500 mgo
p o ein was injec ed (20). A e 2 weeks, 50 ml o abbi blood was collec ed and
se um was sepa a ed by le ing i coagula e o e nigh a 4°C and hen cen i ug-
ing i . The ob ained se um, con aining monospeci ic an i-GAPDH2 polyclonal
an ibodies, was sampled and s o ed a 220°C.
Immunoblo (Wes e n blo ) assays o p o ein samples we e ca ied ou a e
elec opho esis in SDS–12% polyac ylamide slab gels. P o eins we e elec oblo -
ed on o a ni ocellulose memb ane (Bio-Rad) by using a Biome a Fas -Blo
B32 cell (B. B aun Bio ech, Melsungen, Ge many) and incuba ed wi h 1:250- old
dilu ed an ise um in T is-bu e ed saline (TBS) wi h 5% (w / ol) skim milk. The
memb ane was hen washed ou imes (15 min each) in TBS plus 0.05% ( ol/ ol)
Tween 20 (TBS ) and incuba ed o 2 h wi h a goa an i- abbi immunoglobulin
G an ibody-pe oxidase conjuga e (1:1,000; Boeh inge Mannheim). A e ou
15-min ounds o washing wi h TBS , he ni ocellulose il e was de eloped
unde a mix u e o TBS, 2 mM H
2
O
2
, and 10 mM 4-chlo o-3-naph hol in
me hanol. Fil e s we e p ocessed and when necessa y quan i ied wi h an analy -
ical imaging ins umen (Bio Image; Millipo e).
Nucleo ide and amino- e minal p o ein sequence accession numbe s. The
EMBL/GenBank da abase accession numbe o he Synechocys is sp. s ain PCC
6803 DNA agmen con aining he gap-2 gene is X83564. The accession numbe
in he SwissP o da abase o bo h na u al and ecombinan GAPDH2 amino-
e minal p o ein sequences is P80506.
RESULTS AND DISCUSSION
Func ional complemen a ion o a gap E. coli mu an by he
gap-2 gene o Synechocys is sp. s ain PCC 6803. Plasmids con-
aining a o al genomic DNA lib a y o Synechocys is sp. s ain
PCC 6803 cons uc ed on he ClaI si e o he pBS SK
1
ec o
we e used o ans o m he gap E. coli mu an DF221. Plasmid
ex ac ion om clones g own in liquid LB medium supplied
wi h ampicillin indica ed ha a unique 10.6-kb plasmid (pFV1)
was p esen in he complemen ed mu an . pFV1 was subcloned
un il a plasmid o ca. 5.9 kb con aining a 3-kb KpnI-D aI inse
(pFV8), s ill e aining he capaci y o complemen he gap
mu a ion, was cons uc ed. As shown in Table 1, whe eas he
NAD-dependen ac i i y o he ypical glycoly ic enzyme was
ound in c ude ex ac s o wild- ype E. coli s ains like K-12, no
signi ican GAPDH ac i i y was de ec ed in he gap E. coli
mu an s. In con as , c ude ex ac s om he complemen ed
gap mu an s ha bo ing plasmid pFV8 also exhibi ed signi ican
le els o NADP-dependen ac i i y, he alues ob ained wi h
bo h py idine nucleo ides being simila , a dis inc i e ea u e o
Synechocys is sp. s ain PCC 6803 cell ex ac s. These esul s
s ongly sugges ed ha he cyanobac e ial NAD(P)-dependen
GAPDH2, and no he s ic ly NAD-dependen glycoly ic
TABLE 1. NAD(P)-dependen GAPDH speci ic ac i i ies o cell
ex ac s o Synechocys is sp. s ain PCC 6803 and di e en E. coli
s ains and g ow h abili y o E. coli in di e en media
O ganism
GAPDH sp ac (U/mg
o p o ein)
a
G ow h on M63
supplemen ed wi h
b
:
NAD
dependen NADP
dependen Succina e plus
glyce ol Glucose
Synechocys is sp. s ain
PCC 6803 0.048 0.049 NA NA
E. coli K-12 (wild
ype) 0.860 ,0.001
c
111 111
E. coli DF221 (gap,
Am mu a ion) ,0.001 ,0.001 12
E. coli W3CG (gap
gap-1::Tn10 Tc
),0.001 ,0.001 11 2
E. coli DF221(pFV8) 0.601 0.610 111
E. coli W3CG(pFV8) 6.602 6.525 11 111
a
E. coli ac i i y was de e mined in glucose-g own cul u es excep o gap
mu an s. Means o a leas h ee di e en expe imen s a e p esen ed; he s an-
da d e o was less han 15% in all cases.
b
Only o E. coli s ains. G ow h pheno ypes we e sco ed as ollows: 111,
apid g ow h (;75-min doubling ime); 11, mode a e g ow h (;105-min dou-
bling ime); 1, slow g ow h (;140-min doubling ime); 2, no de ec able g ow h.
NA, no applicable.
c
Negligible alues (below he de ec ion limi ).
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GADPH o he en e obac e ium, was he unc ionally ex-
p essed enzyme in he ecombinan clones. The g owing ca-
paci y in LB medium o he E. coli W3CG s ain ha bo ing
plasmid pFV8 was simila o ha o wild- ype s ain K-12 o
DH5a(Table 1), sugges ing an e icien pe o mance o he
Synechocys is sp. s ain PCC 6803 enzyme in deg ada i e ca -
bon me abolism o he en e obac e ium. The inding ha cell
ex ac s o he ecombinan clones exhibi ed wi h bo h py idine
nucleo ide co ac o s speci ic ac i i y le els 10- o 100- old
highe han hose ound in Synechocys is sp. s ain PCC 6803
indica ed ha GAPDH2 was o e exp essed. Likewise, whe eas
no band clea ly a ibu able o he o e exp essed GAPDH
subuni was de ec ed upon SDS-polyac ylamide gel elec o-
pho esis (PAGE) o o al p o ein p epa a ions o he W3CG
mu an , a p ominen p o ein band o ca. 38 kDa could be seen
in ex ac s om he complemen ed clones (Fig. 1A), i s appa -
en size being sligh ly g ea e han ha o he ypical glycoly ic
enzyme subuni (36 kDa) bu i ing well wi h ha o he
pu i ied Synechocys is sp. s ain PCC 6803 GAPDH2 (see be-
low). The ecombinan GAPDH2 subuni was also immuno-
de ec ed in Wes e n blo analysis o p o ein ex ac s om he
ecombinan E. coli clones, using hese an i-GAPDH2 an ibod-
ies (Fig. 1B), hus demons a ing unambiguously he he e ol-
ogous o e exp ession o he Synechocys is sp. s ain PCC 6803
enzyme.
The unc ional complemen a ion o E. coli gap mu an s,
widely applied o clone gap genes coding o NAD-dependen
glycoly ic GAPDHs om he e o ophic eubac e ia (4, 11, 51),
has been success ully used o clone a Synechocys is sp. s ain
PCC 6803 gap-2 gene in ol ed in pho osyn he ic ca bon assim-
ila ion. This sugges s ha Synechocys is sp. s ain PCC 6803
GAPDH2 should be a bi unc ional enzyme ha , in addi ion o
ope a ing in he RPP pa hway in pho osyn hesis, also unc ions
e icien ly in glycolysis, he main physiological ole o GAPDH
in he e o ophic eubac e ia. The e o e, he po en ial p oduc s
o he gapC-like gene gap-1 o gap-3, ecen ly ound in some
cyanobac e ial genomes (8, 28), could in p inciple no be nec-
essa y o ul ill he me abolic demands o a ca abolic NAD-
dependen GAPDH in hese pho osyn he ic p oka yo es. In
ac , Synechocys is sp. s ain PCC 6803 GAPDH2 is de ec ed
and gap-2 is exp essed a high le els unde all me abolical
egimens es ed by us (see below), while he o he possible gap
p oduc in his cyanobac e ium, i.e., ha o gap-1, is ei he
ba ely de ec able o no obse ed a all (49). Addi ional e i-
dence sugges ing ha GAPDH2 should be he only unc ional
GAPDH was ob ained om immuno i a ion expe imen s wi h
he an i-GAPDH2 an ibody, since comple e inhibi ion o bo h
NAD- and NADP-dependen ac i i ies was achie ed in o al
p o ein ex ac s om Synechocys is sp. s ain PCC 6803 (see
below).
The whole Synechocys is sp. s ain PCC 6803 DNA inse
con ained in plasmid pFV8 (a o al o 2,984 bp) was comple ely
sequenced in bo h s ands. Since only one comple e open ead-
ing ame (ORF) o 1,011 bp, a ound he size expec ed o a
gap gene, was ound in he inse , we p esumed ha i co e-
sponded o he gap-2 gene coding o Synechocys is sp. s ain
PCC 6803 GAPDH2. This was la e con i med by a simila i y
sea ch using he FASTA p og am (18a) agains he ecen ly
published en i e genomic sequence o Synechocys is sp. s ain
PCC 6803 (25). The gap-2 coding sequence ound in he pFV8
inse showed 99.5% iden i y wi h he ORF sll1342 (posi ions
3279135 o 3278122) assigned o a gap-2 gene (25), hus expe -
imen ally alida ing his ini ial assignmen . The 337-amino-
acid (aa) p edic ed GAPDH2 p o eins we e i ually iden ical
(100% simila i y) since he i e obse ed nucleo ide changes
p oduced ei he silen o conse a i e amino acid changes ( wo
G o A and one I o F). Nucleo ide sequences o he lanking
egions o gap-2 in he pFV8 inse we e also consis en wi h
he con ex o his gene in he Synechocys is sp. s ain PCC 6803
genome: (i) a unca ed (1,376-bp) ORF s a ing 199 bp up-
s eam gap-2 showed 99.5% iden i y o he 59 egion o ape-2
(sll1343), a gene po en ially coding an 869-aa aminopep idase,
and (ii) a noncoding 255-bp sequence sepa a ed he 59end o
gap-2 om ano he unca ed ORF (145 bp) loca ed in he
complemen a y s and, which was 100% iden ical o he 59
egion o he gene (mu C, sl 1423) likely encoding a 505-aa
N-ace ylmu ama ealanine ligase (25). The s a codon o gap-2
was iden i ied as GTG, a ai ly common ea u e o Synecho-
cys is sp. s ain PCC 6803 genes (25); his iden i ica ion was
la e con i med by di ec analysis o he amino- e minal se-
quence o he GAPDH2 p o ein (see below). A po en ial
Shine-Dalga no sequence (59-GAGG-39) was ound 6 bp up-
s eam o he gap-2 ini ia ion codon. The ansc ip ional s a
poin was de e mined by p ime ex ension analysis o he up-
s eam egion o gap-2 (Fig. 2) o be an A loca ed 48 bp
ups eam o he ini ia ion codon; 10 bp ups eam, a 59-TATG
AT-39sequence ha could ac as a pu a i e 210 egion was
ound. No u he ups eam 235 egion seems o exis , as is
common among cyanobac e ial genes (14). The noncoding
199-bp downs eam egion o gap-2 con ained an in e ed e-
pea ollowed by an oligo(T) ac ound 74 bp om he ans-
la ional e mina ion codon TAG. The DGo he po en ial
s em-loop s uc u e o med by his in e ed epea , calcula ed
by he me hod o F eie e al. (17), is 242.2 kJ/mol. These
ea u es a e sugges i e o a ac o -independen eubac e ial
ansc ip ion e mina o . O e all, hese da a sugges ed ha he
ansc ip o Synechocys is sp. s ain PCC 6803 gap-2 should be
FIG. 1. (A) Coomassie blue-s ained SDS-PAGE elec opho e og am show-
ing he p o ein pa e ns o cell ex ac s om se e al E. coli s ains g own in
di e en media. Lane a, wild- ype K-12 s ain in minimal medium M63 plus
glucose; lane b, DF221 gap mu an in M63 plus succina e and glyce ol; lane c,
W3CG gap mu an ans o med wi h pFV8 plasmid in M63 plus glucose and
ampicillin; lane d, W3CG mu an in M63 plus succina e and glyce ol; lane e,
W3CG mu an ans o med wi h plasmid pBS SK
1
in M63 plus succina e, glyc-
e ol, and ampicillin. The a ow ma ks he posi ion o he o e exp essed 38-kDa
ecombinan Synechocys is sp. s ain PCC 6803 GAPDH2 p o ein in he cell
ex ac co esponding o lane c. (B) Immunoblo analysis wi h monospeci ic
an ibodies aised agains Synechocys is sp. s ain PCC 6803 GAPDH2 o he cell
ex ac s used o panel A. Abou 70 mg o p o ein was loaded pe lane in all cases.
The posi ions and molecula masses o ma ke p o eins a e indica ed.
4516 VALVERDE ET AL. J. BACTERIOL.
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monocis onic and ha e a size o ca. 1.2 kb, as was u he
demons a ed by No he n blo analysis (see below). The cod-
ing sequence o gap-2 was in e sely o ien ed wi h espec o he
ec o p omo e in he inse o plasmid pFV8 and should
he e o e be ansc ibed om i s own p omo e elemen s in
he complemen ed E. coli clones. I should be no ed ha he
o he gap gene ound in he Synechocys is sp. s ain PCC 6803
genome (gap-1, which belongs o he highly conse ed gapC
supe amily [28]) is loca ed in a comple ely di e en egion o
he Synechocys is sp. s ain PCC 6803 genome (sl 0884; posi-
ions 1147034 o 1148098) (25). I s deduced amino acid se-
quence has only abou 50% iden i y wi h he p edic ed se-
quence o GAPDH2 o Synechocys is sp. s ain PCC 6803.
The nucleo ide sequence o Synechocys is sp. s ain PCC
6803 gap-2 has high iden i y, in he ange o 70 o 80%, wi h
wo o he cyanobac e ial gap-2 genes, hose o he cyanobac-
e ia Anabaena a iabilis ATCC 29413 (28) and Synechococcus
sp. s ain PCC 7942 (48) ecen ly epo ed, and wi h gapA and
-Bgenes om highe plan s (8, 42) bu only abou 50% iden i y
wi h he gapC-like genes gap-1 and gap-3 om hose cyanobac-
e ia (28) as well as wi h ypical glycoly ic gapC genes om
o he eubac e ia, plan s, and animals (16). The deduced amino
acid sequence o he Synechocys is sp. s ain PCC 6803
GAPDH2 displayed high iden i y (ca. 80%) wi h hose o he
chlo oplas ic NADP-dependen GAPDHs encoded by gapA
and -Bgenes in highe plan s (8, 42) and gapA genes in eu-
ka yo ic algae (8, 26, 28). Compa ison wi h he amino acid
sequences deduced om he di e en gap genes ecen ly e-
po ed o o he cyanobac e ia, namely, hose o A. a iabilis
(28) and Synechococcus sp. s ain PCC 7942 (48), showed high
iden i y (ca. 75%) wi h hose o he gap-2 genes and much
lowe iden i y wi h he gapC-like genes gap-1 and gap-3 (60 and
45%, espec i ely). The o e all iden i ies wi h he deduced
amino acid sequences o glycoly ic GAPDHs om o he eu-
bac e ia, plan s, o animals we e a ound 50%. I should be
no ed ha he amino- e minal amino acid sequence de e -
mined by he Edman deg ada ion me hod o GAPDH2 pu i-
ied om Synechocys is sp. s ain PCC 6803 cells (see below)
ma ches well wi h he sequence deduced om he cloned Syn-
echocys is sp. s ain PCC 6803 gap-2 gene and is simila o ha
epo ed o he Synechococcus sp. s ain PCC 7942 GAPDH2
p o ein (48). The high deg ee o iden i y o he gap-2 gene
cloned by unc ional complemen a ion wi h gapA and gapB,
which code o plan chlo oplas ic GAPDHs, is consis en wi h
he ole o i s p oduc in pho osyn he ic ca bon assimila ion in
Synechocys is sp. s ain PCC 6803.
The amino acid sequence deduced om he gap-2 gene o
Synechocys is sp. s ain PCC 6803 has been analyzed and ound
o ha e he s uc u al ea u es ypical o o he gap genes, in-
cluding (i) he well-conse ed sequence o he ac i e si e
152
ASCTTNCL
159
(acco ding o he numbe ing o he p e-
dic ed Synechocys is sp. s ain PCC 6803 GAPDH2) a ound he
C
154
(in bold ace) ha o ms he hioes e bond wi h he sub-
s a e in he ca alysis (30) and (ii) he so-called S-loop egion
( esidues 183 o 205) which is in ol ed in he in e ac ion be-
ween subuni s and he binding o he nucleo ide co ac o and
displayed p oka yo ic signa u es (16). A p oline esidue o his
egion p esen in glycoly ic GAPDHs, P
188
acco ding o he
numbe ing o Bacillus s ea o he mophilus GAPDH (5), has
been pos ula ed o be in ol ed in py idine nucleo ide co ac o
speci ici y con e ing absolu e speci ici y o NAD (12). No e-
wo hy, his esidue is eplaced wi h a se ine, S
193
, in he
Synechocys is sp. s ain PCC 6803 GAPDH2 amino acid se-
quence; hus, he enzyme is able o use NADP as a co ac o , as
is also he case o all gapA/B-encoded chlo oplas ic GADPHs
so a s udied (7, 16). Since, in con as wi h hese enzymes, he
cyanobac e ial GAPDH2 is able o use bo h py idine nucleo-
ides e icien ly (chlo oplas ic GAPDHs ha e only ma ginal
ac i i y wi h NAD), o he amino acid esidues would p obably
be in ol ed in he deg ee o NADP/NAD disc imina ion by he
GAPDHs implica ed in pho osyn he ic ca bon assimila ion in
di e en o ganisms. The GAPDH2 om Synechocys is sp.
s ain PCC 6803 has i e C esidues, wo o which (C
154
and
C
158
, as desc ibed abo e) a e clus e ed in a egion di ec ly
in ol ed in ca alysis (30); he o he s (C
19
,C
75
, and C
292
) a e
sp ead o e he p o ein sequence. These h ee C esidues a e,
howe e , p obably no in ol ed in educ i e enzyme ac i a ion
since i has been epo ed ha he NADP-dependen GAPDH
ac i i y o Synechocys is sp. s ain PCC 6803 cell ex ac s is no
ac i a ed ei he in i o by ligh o in i o by hiol- educing
eagen s like DTT (31, 47), as we ha e con i med o bo h
c ude and pu e p epa a ions o na i e and ecombinan Syn-
echocys is sp. s ain PCC 6803 GAPDH2 (da a no shown). All
o he ca aly ic esidues p oposed o be in ol ed in he
GAPDH enzyma ic mechanism (30) (H
181
,T
184
, and R
234
) a e
also p esen in he amino acid sequence deduced om Syn-
echocys is sp. s ain PCC 6803 gap-2.
P ope ies o pu i ied na u al and ecombinan GAPDH2 o
Synechocys is sp. s ain PCC 6803. The NAD(P)-dependen
GAPDH2 has been pu i ied abou 2,000- old o elec o-
pho e ic homogenei y om pho oau o ophically g own Syn-
echocys is sp. s ain PCC 6803 cells wi h a yield o ca. 30%,
using a p ocedu e in ol ing con en ional ammonium sul a e
ac iona ion, anion-exchange ch oma og aphy, hyd ophobic
ch oma og aphy, and dye-ligand ch oma og aphy. The p o ein
analysis by SDS-PAGE o he p epa a ions ob ained a e he
di e en pu i ica ion s eps demons a ed he homogenei y o
he inal GAPDH2 p epa a ion, which exhibi ed high speci ic
ac i i y alues, ypically ca. 120 U/mg. A single p o ein band
wi h a molecula mass o ca. 38 kDa was seen in he elec o-
pho e og am o he inal enzyme p epa a ion (Fig. 3B). The
ecombinan Synechocys is sp. s ain PCC 6803 GAPDH2 was
also pu i ied o elec opho e ic homogenei y (abou 30- old)
om E. coli W3CG cells ha bo ing pFV8 by using a pu i ica-
ion p ocedu e simila o ha used o he na u al enzyme. A
single p o ein band o 38 kDa, indis inguishable om ha o
he na u al GAPDH2 and wi h a subuni size sligh ly g ea e
han ha o he ypical glycoly ic GAPDH, was also ound in
he inal p epa a ion on SDS-polyac ylamide gels (Fig. 4A). I
should be no ed ha bo h NAD- and NADP-linked ac i i ies
we e moni o ed h oughou he comple e pu i ica ion o na -
u al and ecombinan Synechocys is sp. s ain PCC 6803
FIG. 2. Mapping o he GAPDH2 p esumed ansc ip ional s a poin by
p ime ex ension analysis o o al RNA pu i ied om pho oau ophically g own
Synechocys is sp. s ain PCC 6803 cells. The ex ension p oduc s we e loaded in o
he lane on he le . The DNA sequence a he igh co esponds o he egion
o he empla e s and o gap-2 a ound he wo T bases (bold ace) iden i ied as
ansc ip ion s a si es.
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GAPDH2, and a pe ec o e lapping as a single symme ical
peak was obse ed du ing all ch oma og aphic s eps (Fig. 3A).
We compa ed pu i ied na u al and ecombinan Synechocys-
is sp. s ain PCC 6803 GAPDH2 wi h ega d o a ious s uc-
u al and kine ic p ope ies and ound no signi ican di e -
ences be ween hem. Thus, hey had an homo e ame ic
s uc u e in he na i e s a e as sugges ed by he i ually iden-
ical molecula masses, ca. 160 65 kDa, de e mined by FPLC
gel il a ion. Mo eo e , he wo enzymes exhibi ed he same
beha io in nondena u ing glyce ol-polyac ylamide g adien
gels (es ima ed molecula mass by his me hod, ca. 155 65
kDa) as well as he same pI alue (ca. 4.85 60.1) upon ei he
column ch oma o ocusing o isoelec ic ocusing, indica ing
iden ical su ace cha ge dis ibu ion and he e o e no signi i-
can con o ma ional di e ences be ween hem. The na u al
and ecombinan Synechocys is sp. s ain PCC 6803 GAPDH2s
we e also loca ed in he nondena u ing polyac ylamide gels by
in si u s aining o dehyd ogenase ac i i y using e azolium
dyes wi h ei he NAD o NADP as he co ac o ; he ac i i y
bands showed simila mig a ion pa e ns (Fig. 3C). The amino-
e minal ends o bo h na u al (TRVAINGFGR) and ecom-
binan (MTRVAINGFGR) Synechocys is sp. s ain PCC 6803
GAPDH2s we e sequenced by he Edman deg ada ion
me hod. The i ual iden i y o he ob ained amino acid se-
quences sugges ed no di e ences in p ima y p o ein s uc u e
be ween hem. Howe e , i is no ewo hy ha while in he
na u al enzyme pu i ied om he cyanobac e ium he ini ial
me hionine esidue was emo ed, he ecombinan p o ein s ill
e ained he s a ing amino acid deduced om he gap-2 gene.
This could be due o a less e icien ma u a ion mechanism in
he E. coli hos o o an excess o p o ein p oduc o which
comple e ma u a ion had no been possible.
Bo h pu i ied na u al and ecombinan Synechocys is sp.
s ain PCC 6803 GAPDH2s we e able o use NAD o NADP
wi h i ually he same e iciency; speci ic ac i i ies o ca. 120
U/mg, simila o hose o o he GAPDHs (7, 16), we e ound
in he pu i ied p epa a ions. The op imal pH alue o he
oxida i e eac ion was in bo h cases 8.2 60.1, wi h conside -
able ac i i y in he ange o 7 o 10, and he appa en op imal
empe a u e was 35 62°C. Appa en K
m
and V
max
we e mea-
su ed o bo h NAD and NADP in his eac ion, and iden ical
FIG. 3. (A) P o ein and G3P dehyd ogenase ac i i y elu ion by a double
linea g adien o dec easing ammonium sul a e concen a ion and inc easing
concen a ion o glyce ol du ing hyd ophobic in e ac ion ch oma og aphy on
phenyl-Sepha ose HP, a pu i ica ion s ep o GAPDH2 om pho oau o ophi-
cally g own Synechocys is sp. s ain PCC 6803 cells. Bo h NAD- and NADP-
dependen ac i i ies elu ed simul aneously as pe ec ly o e lapped symme ical
peaks. (B) Coomassie blue-s ained SDS-PAGE elec opho e og am showing
di e en Synechocys is sp. s ain PCC 6803 GAPDH2 pu i ica ion s ep ac ions.
Lane a, 40 o 88% ammonium sul a e p o ein ac ion; lane b, anion-exchange
ch oma og aphy elua e pool; lane c, hyd ophobic-in e ac ion ch oma og aphy
elua e pool; lane d, pu e p o ein p epa a ion a e dye-ligand ch oma og aphy.
Aliquo s o p o ein p epa a ions con aining ei he 50 mg (lanes a and b) o 20 mg
(lanes c and d) o p o ein we e applied. The a ow poin s o he 38-kDa
GAPDH2 subuni band. (C) Localiza ion, wi h ei he NADP o NAD, o G3P
dehyd ogenase ac i i y o pu i ied na u al Synechocys is sp. s ain PCC 6803
GAPDH2 a e PAGE unde nondena u ing condi ions. Ac i i y s aining was
pe o med in si u in he glyce ol-polyac ylamide g adien gels incuba ed wi h
eac ion mix u es con aining ei he NADP (le ) o NAD ( igh ) and de eloped
wi h p-ni oblue e azolium chlo ide. FIG. 4. (A) Coomassie blue-s ained SDS-PAGE elec opho e og am show-
ing pu i ied p epa a ions o di e en GAPDHs. Lane a, pu i ied na u al Syn-
echocys is sp. s ain PCC 6803 GAPDH2 p o ein; lane b, ecombinan Synecho-
cys is sp. s ain PCC 6803 GAPDH2 p o ein pu i ied om pFV8- ans o med
W3CG gap mu an ; lane c, a ypical glycoly ic GAPDH om mammalian issue
(skele al muscle o Jaculus o ien alis). Aliquo s o p o ein p epa a ions con ain-
ing 10 mg o pu i ied enzymes we e applied. The hick a ow ma ks he posi ion
o he 38-kDa GAPDH2 subuni . No e he lowe molecula mass (36 kDa) o he
mammalian glycoly ic GAPDH subuni ( hin a ow). (B) Immunoblo analysis o
cell- ee p o ein ex ac s om di e en o ganisms using monospeci ic an ibodies
aised agains Synechocys is sp. s ain PCC 6803 GAPDH2. Lane a, E. coli W3CG
ans o med wi h pFV8; lane b, wild- ype E. coli s ain K-12; lane c, Synechocys is
sp. s ain PCC 6803; lane d, E. coli W3CG bea ing a pBS SK
1
de i a i e plasmid
con aining he gap-1 gene om Anabaena sp. s ain PCC 7120 and o e exp ess-
ing GAPDH1; lane e, Anabaena sp. s ain PCC 7120; lane , An i hinum majus;
lane g, Spinacea ole acea; lane h, Chlamydomonas einha d ii 6145c (a chlo o-
phycean mic oalga); lane i, Po phy idium pu pu eum SAG 1380-1a (a hodo-
phycean mic oalga). Abou 70 mg o o al p o ein was loaded pe lane. No e ha
an i-GAPDH2 an ibodies ecognized in highe plan ex ac s bo h gapA and
gapB p o ein p oduc s (ca. 38 and 41 kDa, espec i ely) which cons i u e he
he e o e ame ic chlo oplas ic GAPDHs o hese o ganisms (lanes and g).
4518 VALVERDE ET AL. J. BACTERIOL.
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alues wi hin he expe imen al e o s (30 65mM and 7 61
mmol/min, espec i ely) we e es ima ed o bo h coenzymes,
using he na u al and ecombinan GAPDH2s. These alues
a e, mo eo e , e y simila o hose desc ibed o o he cy o-
solic and chlo oplas ic GAPDHs (7), a cha ac e is ic o hese
enzymes being a ypical subs a e inhibi ion, which we ook
in o conside a ion when calcula ing he appa en kine ic pa-
ame e s o py idine nucleo ides. The kine ic pa ame e s o
G3P es ima ed o bo h p o eins we e also alike wi h ei he
NAD o NADP as he co ac o (K
m
, 270 630 mM; V
max
,176
2mmol/min), again wi h i ually no di e ences be ween e-
combinan and na u al GAPDHs, clea ly indica ing ha he
wo should be unc ionally iden ical. Bo h enzymes we e also
e y speci ic o he phospho yla ed subs a es, no signi ican
ac i i y being obse ed wi h o he phospho yla ed o nonphos-
pho yla ed ioses, like dihyd oxyace one-phospha e, e en in
he millimola concen a ion ange (da a no shown). On he
o he hand, he appa en K
m
alues es ima ed o NADPH and
NADH in he educ i e eac ion ( o ma ion o G3P om 1,3-
bisphosphoglyce a e) we e also i ually iden ical, 10 61mM,
bu he V
max
alues we e di e en , 2.2 60.3 mmol/min o
NADPH and 4.5 60.4 mmol/min o NADH, indica ing a
highe ca aly ic e iciency o NADH han o NADPH. In ac ,
his is he only kine ic di e ence ound be ween bo h py idine
nucleo ides in Synechocys is sp. s ain PCC 6803 GAPDH2. I
should be no ed ha his esul is qui e di e en om wha has
been epo ed o he chlo oplas ic GAPDH o highe plan s,
which is p ac ically NADP speci ic, i s a ini y o he phos-
pho yla ed coenzyme being much highe (ca. 100- old) (7, 16).
To ou knowledge, an es e ase ac i i y has been desc ibed so
a only o he classical glycoly ic NAD-dependen GAPDH
(44). We ound ha Synechocys is sp. s ain PCC 6803
GAPDH2 also exhibi ed a high hyd olase ac i i y (speci ic ac-
i i ies o pu i ied na u al and ecombinan enzymes in he
same ange, 200 o 250 U/mg) when we used one o a a ie y
o o ganic p-ni ophenyles e s (ei he p-ni ophenylace a e, p-
ni ophenylbu y a e o p-ni ophenylphospha e) as he sub-
s a e. The physiological signi icance o he es e ase ac i i y o
cyanobac e ial GAPDH2 emains o be in es iga ed.
Wes e n and No he n blo analysis o gap-2 exp ession in
Synechocys is sp. s ain PCC 6803. Monospeci ic polyclonal
an ibodies aised in a abbi agains he elec opho e ically
pu e na u al Synechocys is sp. s ain PCC 6803 GAPDH2 we e
used o analyze he exp ession o he gap-2 gene a he p o ein
le el. These an ibodies ecognized in Wes e n blo s a single
p o ein band o ca. 38 kDa, co esponding o he GAPDH2
subuni , in p o ein ex ac s ei he om his cyanobac e ium o
om E. coli W3CG bea ing pFV8 (Fig. 4B; see also Fig. 1B).
Immuno i a ion expe imen s showed ha hese an ibodies
we e able o comple ely inhibi bo h NAD- and NADP-depen-
den GAPDH ac i i ies in o al p o ein ex ac s om Synecho-
cys is sp. s ain PCC 6803 cells g own unde ei he pho oau-
ophic o he e o ophic condi ions, indica ing ha he
GAPDH2 should be he only enzyme wi h such ac i i ies
p esen in hese p epa a ions (da a no shown). C oss- eac ion
s udies p o ided in o ma ion on he possible s uc u al and
phylogene ic ela ionships o he Synechocys is sp. s ain PCC
6803 GAPDH2 wi h homologous enzymes om di e se o gan-
isms (Fig. 4B). The an ibodies we e able o ecognize no only
he GAPDH2s o o he cyanobac e ia bu also he chlo oplas-
ic NADP-dependen GAPDHs o plan s and a a ie y o eu-
ka yo ic mic oalgae. A 38-kDa p o ein band was ound in
Wes e n blo s o cell ex ac s om all o he cyanobac e ia
es ed and om euka yo ic mic oalgae, whe eas wo clea
bands, co esponding o gapA and gapB p oduc s, could be
seen in highe plan s. No bands we e immunode ec ed in cell
ex ac s con aining only he glycoly ic GAPDH, such as hose
om E. coli K-12 and DH5a(Fig. 1B) o om nonpho osyn-
he ic p o is s, such as he cilia ed p o ozoa Te ahymena py i-
o mis and Pa amecium e au elia, and di e se mammalian
issues (da a no shown). The ecombinan GAPDH1 (an
NAD-speci ic cyanobac e ial enzyme encoded by he gapC-like
gene gap-1)o Anabaena sp. s ain PCC 7120 (49) also was no
ecognized by hese an ibodies. I seems, he e o e, ha p od-
uc s o bo h he cyanobac e ial gap-2 and he gapA and -B
genes o pho osyn he ic euka yo es sha e s uc u al cha ac e -
is ics ha a e no ound in he p oduc s o he ubiqui ous gapC
supe amily (8, 21).
The highly speci ic an ibodies o he cyanobac e ial
GAPDH2 allowed us o s udy he exp ession o gap-2 a he
p o ein le el in Synechocys is sp. s ain PCC 6803 wi hou he
possible in e e ence o o he GAPDHs ha in p inciple migh
ha e been p esen in he c ude p epa a ions (Fig. 5A). On he
o he hand, o analyze he exp ession o gap-2 a he mRNA
FIG. 5. Compa a i e analysis o GAPDH2 p o ein and gap-2 ansc ip le els
in Synechocys is sp. s ain PCC 6803 cells g own unde di e en ophic egimens.
(A) Immunoblo s o p o ein ex ac s om Synechocys is sp. s ain PCC 6803,
using he an i-GAPDH2 an ibodies as o Fig. 4B. Lane a, ni a e/ligh ; lane b,
ammonium/ligh ; lane c, ni a e plus glucose/ligh ; lane d, N s a a ion/ligh ; lane
e, ni a e plus glucose/da kness; lane , P s a a ion/ligh . Molecula size ma ke s
we e elec opho esed in he same gel and s ained o p o ein p esence. Abou 70
mg o o al p o ein was loaded pe lane. (B) No he n blo s o o al RNA isola ed
om cells g own in he condi ions desc ibed o panel A. Ei he a 0.94-kb
in e nal gap-2 agmen gene a ed by PCR om plasmid pFV8 ( op) o an
in e nal agmen o npB which codes o Synechocys is sp. s ain PCC 6803
RNase P RNA (loading con ol; bo om) was used as he p obe. The a ow shows
he posi ion o he 1.2-kb gap-2 ansc ip . RNA s anda ds we e elec opho esed
in he same gel and s ained wi h e hidium b omide. Abou 15 mg o o al RNA
was loaded pe lane. (C) Co ela ion o he ela i e le els o enzyma ic ac i i y
and he gap-2 p oduc s, GAPDH2 p o ein, and gap-2 mRNA ansc ip . These
pa ame e s we e quan i ied in Synechocys is sp. s ain PCC 6803 cell ex ac s
om cells g own unde he condi ions speci ied o panel A. Da a a e means o
h ee independen de e mina ions and a e exp essed as pe cen ela i e uni s,
he alue o cells g own wi h ni a e being conside ed 100%. Open ba s, speci ic
ac i i y (100%, 50 63 mU/mg o p o ein); s ippled ba s, quan i a ion o he
immunos ained GAPDH2 p o ein; closed ba s, quan i a ion o he 1.2-kb gap-2
ansc ip RNA band a e no maliza ion wi h he loading con ol.
VOL. 179, 1997 CHARACTERIZATION OF SYNECHOCYSTIS GAPDH2 4519
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le el, a 0.94-kb in e nal agmen o Synechocys is sp. s ain
PCC 6803 gap-2 was used as a p obe in No he n blo analysis
o o al RNA samples isola ed om cul u es o his cyanobac-
e ium; a single ansc ip o abou 1.2 kb, he expec ed size o
he gap-2 p oduc , was ound in all cases (Fig. 5B). The RNA
componen o he ibozyme RNase P, a housekeeping enzyme
in Synechocys is sp. s ain PCC 6803 (50), was used as a loading
con ol. Bo h Wes e n and No he n blo analyses clea ly in-
dica ed ha he exp ession o Synechocys is sp. s ain PCC
6803 gap-2 was a ec ed by he ophic condi ions o he cell
cul u es. A good co ela ion was ound among speci ic ac i i y
alues and immunode ec ed GAPDH2 p o ein and gap-2 an-
sc ip le els in he di e en cul u e condi ions es ed (Fig. 5C).
The main e ec was he ma ked dec ease in all pa ame e s up
o ca. 10% o he con ol alues (pho oau ophic cells) by he
addi ion o glucose, a suga me abolizable by his cyanobac e-
ium (1, 15), ei he in he ligh (pho ohe e o ophy) o in
da kness (chemohe e o ophy). I should be no ed ha cells
cul u ed in he ligh wi h glucose and 10 mM DCMU (s ic
pho ohe e o ophy) p esen ed le els simila o hose cul u ed
in he e o ophic condi ions (da a no shown). In con as , he
a he high le els obse ed in pho oau o ophically g own cells
we e sligh ly a ec ed ei he by he sou ce o ino ganic ni ogen
(ca. 30% dec ease when g own wi h ammonia ins ead o ni-
a e) o by nu i ional s ess condi ions such as N s a a ion o
P limi a ion, bu in he las wo condi ions a dec ease o o al
cellula p o ein was also ound. An appa en ac i a ion o he
enzyme was obse ed only in P-deple ed cells. In summa y,
No he n and Wes e n blo s indica e ha gap-2 is su icien ly
exp essed and GAPDH2 appea s in de ec able le els in Syn-
echocys is sp. s ain PCC 6803 cells g owing unde di e en
ophic condi ions, e en in s ic chemohe e o ophy when no
pho osyn hesis is pe o med a all, so ha i seems able o
accomplish by i sel all po en ial me abolic unc ions, wi hou
he need o ano he gap p oduc .
Ou da a on GAPDH2 egula ion shed new ligh on se e al
con adic o y epo s in he ea ly li e a u e conce ning speci ic
ac i i y measu emen s in cell ex ac s o cyanobac e ia (3, 13,
24, 32, 33). The good co ela ion ound be ween speci ic ac i -
i y, GAPDH2 p o ein, and mRNA gap-2 ansc ip le els du -
ing he d ama ic dec ease o hese pa ame e s obse ed a e
glucose addi ion o Synechocys is sp. s ain PCC 6803 cul u es
indica es ha no p ocess o enzyme ac i a ion o inac i a ion is
in ol ed in hese changes. Thus, egula ion by he p esence o
he me abolizable suga seems o be due o changes in he
mRNA le els a he han o GAPDH2 ac i i y modula ion.
Glucose should no be ega ded as a p ope nu ien bu a he
as a oxic compound since i s addi ion o pho oau o ophic
cul u es o Synechocys is sp. s ain PCC 6803 p omo es a i s
a high cell mo ali y, and cul u es adap ed o pho ohe e o o-
phic condi ions exhibi lowe g ow h a es han pho oau o o-
phic cells (1, 15).
The esul s o Synechocys is sp. s ain PCC 6803 a e in sha p
con as wi h ou p e ious obse a ions on he euka yo ic mi-
c oalga Chlo ella usca (40). In he la e o ganism, he p es-
ence o he suga caused a ma ked dec ease o he NADP-
FIG. 6. Occu ence o bo h GAPDH2 and gap-2 in s ains ep esen a i e o
some cyanobac e ial axonomic g oups. (A) Immunode ec ion o he GAPDH2
p o ein in cell ex ac s o di e en cyanobac e ia by Wes e n blo ing using he
an i-GAPDH2 an ibodies aised agains he Synechocys is sp. s ain PCC 6803
enzyme. Abou 70 mg o o al p o ein was loaded pe lane. (B) Sou he n blo
analysis o genomic DNAs om di e se cyanobac e ia diges ed wi h HindIII and
p obed wi h a
32
P- adiolabelled 0.94-kb in e nal gap-2 agmen gene a ed by
PCR om pFV8. The s ains a e iden i ied by hei collec ion numbe s. The
posi ions o EcoRI-HindIII- es ic ed lDNA agmen s used as s anda ds (in
he ange o 21 o 0.5 kb) a e indica ed on he le side.
TABLE 2. NAD(P)-dependen GAPDH speci ic ac i i ies o cell
ex ac s om se e al ep esen a i e s ains o some cyanobac e ial
axonomic g oups
a
Cyanobac e ial s ain
Sp ac (mU/mg o p o ein)
b
NAD/
NADP
ac i i y
a io
NAD
dependen NADP
dependen
NAD plus
NADP in
assay
Ch oococcales
Synechocys is sp.
s ain PCC 6803 50 49 45 1.02
Synechococcus sp.
s ain PCC 7942 14 24 37 0.58
Pleu ocapsales
De moca pa sp.
s ain PCC 7437 63 72 69 0.89
Oscilla o iales
Pseudanabaena sp.
s ain PCC 6903 26 27 22 0.96
Nos ocales
Anabaena sp. s ain
PCC 7120 25 21 23 1.19
Anabaena sp. s ain
ATCC 29413 71 60 58 1.18
Nos oc sp. s ain
PCC 7107 38 31 34 1.22
Calo h ix sp. s ain
PCC 7601 13 11 10 1.18
S igonema ales
Fische ella sp. s ain
UTEX 1829 22 21 19 1.05
a
All cyanobac e ia we e g own pho oau o ophically in BG11 medium (ni-
a e as he ni ogen sou ce) unde con inuous whi e ligh (25 W/m
2
) and bub-
bling wi h ai plus 1.5% ( ol/ ol) CO
2
.
b
Da a a e he means o a leas h ee independen de e mina ions using
la e-exponen ial-phase cul u es; he s anda d e o was less han 15% in all cases.
4520 VALVERDE ET AL. J. BACTERIOL.
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dependen GAPDH ac i i y and p o ein amoun o unde ec able
alues, bu pho ohe e o ophic cul u es exhibi ed e en highe
g ow h a es han he pho oau o ophic ones. Like o he pho o-
syn he ic euka yo es, C. usca has a cy osolic NAD-dependen
GAPDH, di e en om he chlo oplas ic NADP-dependen en-
zyme, ha pa icipa es in he ca abolic pa hways in ol ed in
suga assimila ion (29, 40). The di e en beha io o Synecho-
cys is sp. s ain PCC 6803 migh be due o he capaci y o
glucose o ac in his o ganism as a ca abolic ep esso o he
GAPDH2 in ol ed in pho osyn he ic ca bon assimila ion. In
o he wo ds, i no GAPDH o he han GAPDH2 could e i-
cien ly accomplish ca bohyd a e ca abolism in Synechocys is sp.
s ain PCC 6803, ep ession o gap-2 exp ession would neces-
sa ily lead o an impai men o g ow h unde pho ohe e o o-
phic condi ions, as was ound o be he case (15). O e all, ou
esul s a e in ag eemen wi h he amphibolic ole assigned o
GAPDH2 and sugges ha only his enzyme is unc ional in
his cyanobac e ium unde bo h pho osyn he ic and he e o o-
phic g ow h condi ions.
Since he Synechocys is sp. s ain PCC 6803 GAPDH2 is
de ec ed a conspicuous le els unde bo h pho o ophic and
he e o ophic condi ions, his enzyme could e icien ly unc ion
ei he in he pho osyn he ic RPP pa hway o in ca bohyd a e
ca abolism. Kine ic da a indica e, howe e , ha he Synecho-
cys is sp. s ain PCC 6803 GAPDH2 exhibi s in he educ i e
(anabolic) eac ion a wo old-highe ca aly ic e iciency (V
max
/
K
m
) wi h NADH han wi h NADPH, despi e equal K
m
alues
o bo h py idine nucleo ides. In con as , ypical chlo oplas ic
enzymes a e abou 100 imes mo e e icien wi h NADPH (7).
Thus, GAPDH2 seems in his espec o be somewha ela ed
o GAPDH o pho osyn he ic bac e ia, which pe o m anoxy-
genic pho osyn hesis using NADH as he educing co ac o in
he RPP pa hway (9). Le els o NAD(P)H in cyanobac e ial
cells migh change signi ican ly depending on he en i onmen-
al ophic condi ions, being much highe in ligh -g owing cells,
p o iding enough subs a e o he anabolic eac ions, and low
in da kness due o cyanobac e ial espi a ion (38, 43). I has
been pos ula ed ha he in acellula NAD(P)H/NAD(P) a io
could egula e he cyanobac e ial glucose-6-phospha e and
6-phosphoglucona e dehyd ogenases (43, 45, 46). The possible
esponse o Synechocys is sp. s ain PCC 6803 GAPDH2, which
unlike o he pho osyn he ic GAPDHs seems o p e e NADH
o NADPH in he educ i e eac ion, o changes o hese
me abolic pa ame e s may ha e a egula o y signi icance wo h
in es iga ing.
Occu ence o gap-2 and GAPDH2 among cyanobac e ia.
Genomic DNA and o al p o ein p epa a ions ob ained om
pho oau o ophically g own cells o se e al s ains ep esen a-
i e o some cyanobac e ial axonomic g oups we e es ed o
bo h he gap-2 gene and he GAPDH2 p o ein. The ollowing
s ains we e used o his pu pose: Synechocys is sp. s ain PCC
6803, Synechococcus sp. s ain PCC 7942, De moca pa sp.
s ain PCC 7437, Pseudanabaena sp. s ain PCC 6903,
Anabaena sp. s ains PCC 7120 and ATCC 29413, Nos oc sp.
s ain PCC 7107, Calo h ix sp. s ain PCC 7601, and Fische ella
sp. s ain UTEX 1829. Wes e n blo analysis o he ex ac s
using he an i-GAPDH2 an ibodies clea ly e ealed ha he
p oduc o he gap-2 gene was p esen in all o hem (Fig. 6A),
hus p o ing he gene al exp ession o his gene among cya-
nobac e ia. A single immunos ained p o ein band o 38 kDa
was seen in he p o ein ex ac s om hese s ains, excep in
he case o Synechococcus sp. s ain PCC 7942, which showed
a sligh ly la ge band. This inding is in ag eemen wi h a ecen
epo showing ha his cyanobac e ium p esen s a peculia
GAPDH2 wi h an addi ional amino acid sequence a he ca -
boxy e minus (53). On he o he hand, when ei he a 2.1-kb-
long AseI agmen o he pFV8 inse con aining he Synecho-
cys is sp. s ain PCC 6803 gap-2 gene (no shown) o a 0.94-kb
PCR-gene a ed in e nal agmen o he gene was used as a
p obe in Sou he n blo expe imen s wi h HindIII- es ic ed
cyanobac e ial DNA, clea bands could be de ec ed in all cases
(Fig. 6B), indica ing again a wide dis ibu ion o his gene
among he s ains es ed.
Le els o bo h NAD- and NADP-dependen GAPDH ac i -
i ies we e also de e mined in he o al p o ein ex ac s used in
he expe imen desc ibed abo e. As shown in Table 2, e y
simila le els we e ound o bo h ac i i ies in he di e en
cyanobac e ial ex ac s, alues o a ound 1 being obse ed o
he NAD-/NADP-dependen ac i i y a io, again wi h he e-
ma kable excep ion o Synechococcus sp. s ain PCC 7942, in
which his a io was clea ly lowe . This la e esul could be
expec ed since he GAPDH2 om his cyanobac e ium exhib-
i s lowe a ini y o NAD (53), ano he aspec in which his
enzyme di e s om hose o Synechocys is sp. s ain PCC 6803
and A. a iabilis. No addi i i y be ween bo h ac i i ies ( ha is,
no signi ican inc ease o enzyma ic ac i i y in he p esence o
bo h coenzymes) was obse ed, s ongly sugges ing ha bo h
py idine nucleo ides compe e o he same ac i e si e and ha
only one enzyme is p esen in he ex ac s. I is no ewo hy
ha , once mo e, his was no he case o he peculia
GAPDH2 o Synechococcus sp. s ain PCC 7942. Whe he his
is due ei he o a di e en kind o GAPDH2 egula ion o o
he p esence o ano he NAD-dependen GAPDH emains o
be es ablished. Howe e , al hough Synechococcus sp. s ain
PCC 7942 has wo di e en gapC-like genes, gap-1 and gap-3,
i s peculia GAPDH2 is he only GAPDH enzyme p esen in
c ude ex ac s, as shown by ecen biochemical s udies (48). I
is also in e es ing ha in he pho osyn he ic p o is Cyano-
pho a pa adoxa, a single NAD(P)-dependen GAPDH, also
exhibi ing a clea dual dependence o he nucleo ide co ac o ,
was cha ac e ized in he cyanelle, whe eas he NAD-depen-
den glycoly ic enzyme was ound only in he cy osol (39). Since
his cyanobac e ium-like o ganelle has been pos ula ed as an
e olu iona y elic eminiscen o he ancien cyanobac e ial
endosymbion p ecu so o pho osyn he ic plas ids, hese ind-
ings ag ee wi h ou p oposal on he amphibolic cha ac e o
he cyanobac e ial GAPDH2.
ACKNOWLEDGMENTS
We hank A. Vioque o kindly p o iding he Synechocys is sp. s ain
PCC 6803 genomic lib a y and B. Bachman and A. Plu¨ck ung o he
E. coli gap mu an s DF221 and W3CG, espec i ely. We owe many
hanks o E. Flo es and J. R. Pe´ ez-Cas in˜ei a o commen s on he
manusc ip and W. Lo¨ elha d o he amino- e minal sequencing o
he GAPDH2 p o eins a he Ins i u u¨ Biochemie und Molekula e
Zellbiologie, Uni e si y o Vienna (Aus ia).
This wo k was suppo ed by DGICYT g an s PB-91/0085 and PB-
94/0033 om he Spanish go e nmen and g an PAI CVI-0198 om
he Andalusian go e nmen .
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