APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/99/$04.00⫹0Sep . 1999, p. 3774–3779 Vol. 65, No. 9
Copy igh © 1999, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
Role o N␥-Ace yldiaminobu y a e as an Enzyme S abilize and
an In e media e in he Biosyn hesis o Hyd oxyec oine
DAVID CA
´NOVAS,
1,2
† NUNO BORGES,
2
CARMEN VARGAS,
1
ANTONIO VENTOSA,
1
JOAQUI
´N J. NIETO,
1
*AND HELENA SANTOS
2
Depa amen o de Mic obiologı´a y Pa asi ologı´a, Facul ad de Fa macia, Uni e sidad de Se illa, 41012 Se ille, Spain,
1
and Ins i u o de Tecnologia Quı´mica e Biolo´gica, Uni e sidade No a de Lisboa, 2780 Oei as, Po ugal
2
Recei ed 7 Ap il 1999/Accep ed 8 June 1999
S ain CHR63 is a sal -sensi i e mu an o he mode a ely halophilic wild- ype s ain Halomonas elonga a
DSM 3043 ha is a ec ed in he ec oine syn hase gene (ec C). This s ain accumula es la ge amoun s o
N␥-ace yldiaminobu y a e (NADA), he p ecu so o ec oine (D. Ca´no as, C. Va gas, F. Iglesias-Gue a, L. N.
Csonka, D. Rhodes, A. Ven osa, and J. J. Nie o, J. Biol. Chem. 272:25794–25801, 1997). Hyd oxyec oine,
ec oine, and glucosylglyce a e we e also iden i ied by nuclea magne ic esonance (NMR) as cy oplasmic
o ganic solu es in his mu an . Accumula ion o NADA, hyd oxyec oine, and ec oine was osmo egula ed,
whe eas he le els o glucosylglyce a e dec eased a highe salini ies. The e ec o he g ow h s age on he
accumula ion o solu es was also in es iga ed. NADA was pu i ied om s ain CHR63 and was shown o p o ec
he he molabile enzyme abbi muscle lac a e dehyd ogenase agains he mal inac i a ion. The s abilizing
e ec o NADA was g ea e han he s abilizing e ec o ec oine o po assium diaminobu y a e. A
1
H NMR
analysis o he solu es accumula ed by he wild- ype s ain and mu an s CHR62 (ec A::Tn1732) and CHR63
(ec C::Tn1732) indica ed ha H. elonga a can syn hesize hyd oxyec oine by wo di e en pa hways—di ec ly
om ec oine o ia an al e na i e pa hway ha con e s NADA in o hyd oxyec oine wi hou he in ol emen
o ec oine.
The s a egy de eloped by halophilic and halo ole an bac-
e ia o cope wi h he osmo ic s ess imposed by a hype saline
en i onmen in ol es accumula ion o low-molecula -weigh
o ganic compounds ha a e called compa ible solu es (2).
These compounds can be classi ied in o di e en ca ego ies,
such as polyols and he e osides, suga s, amino acids and hei
de i a i es, N-ace yla ed diamino acids, be aines and e hines,
and ec oines (ec oine and hyd oxyec oine). Recen ly, he e has
been inc eased in e es in compa ible solu es in bio echnology,
since i has been shown ha hese compounds a e able o
p o ec enzymes and whole cells agains s esses, such as he
s esses caused by sal , hea ing, eezing, and desicca ion (8,
11).
The mode a ely halophilic bac e ium Halomonas elonga a
exhibi s one o he wides anges o sal ole ance known; his
o ganism is able o g ow in he p esence o NaCl concen a-
ions anging om ⬃0.1 o ⬃4 M in complex medium (26–28).
Osmoadap a ion is achie ed by accumula ion o glycine be-
aine, which is aken up om he medium o syn hesized om
choline (4, 5), and by syn hesis o ec oine and hyd oxyec oine
(6). The biosyn he ic pa hway o ec oine in his bac e ium has
been elucida ed a he biochemical and gene ic le els (3, 6, 13,
18) (Fig. 1). Ec oine syn hesis occu s in h ee s eps. Fi s ,
aspa a e semialdehyde, an in e media e in amino acid me ab-
olism, is con e ed in o diaminobu y a e (DA), which is sub-
sequen ly ace yla ed o N␥-ace yldiaminobu y a e (NADA).
Cyclic condensa ion o his compound leads o o ma ion o
ec oine (18, 19). Recen ly, Ca´no as e al. isola ed (6) and
cha ac e ized (3) he gene ic egion in ol ed in ec oine syn-
hesis in H. elonga a DSM 3043. This egion comp ises he
ollowing h ee genes: ec A, which encodes he DA ace yl ans-
e ase; ec B, which encodes he DA ansaminase; and ec C,
which encodes he ec oine syn hase (3). In con as o he
well-cha ac e ized ec oine syn hesis pa hway, he biosyn he ic
ou e leading o hyd oxyec oine has no been es ablished ye ,
and he genes in ol ed a e no known. Al hough he e is some
e idence ha hyd oxyec oine could be syn hesized di ec ly
om ec oine (6, 13), his hypo hesis lacks i m suppo .
In a ecen s udy, Ca´no as e al. (6) demons a ed ha
NADA, he immedia e p ecu so o ec oine, plays an osmo-
p o ec i e ole in H. elonga a. In ac , he sal -sensi i e mu an
CHR63 (ec C::Tn1732), which accumula ed NADA, could ol-
e a e highe le els o salini y han he le els ole a ed by mu-
an CHR62 (ec A::Tn1732), which accumula ed DA; his in-
dica ed ha NADA unc ions as a compa ible solu e in mu an
CHR63. This was he i s indica ion ha NADA, which was
disco e ed as a componen o Eupho bia pulche ima la ex
(16), could play a ole in osmo ic adap a ion. In his s udy,
NADA was pu i ied om mu an s ain CHR63 and shown o
s abilize he model enzyme abbi muscle lac a e dehyd oge-
nase (LDH) agains inac i a ion by hea . Mo eo e , accumu-
la ion o solu es was quan i ied as a unc ion o he NaCl
concen a ion in he medium and he g ow h phase. E idence
which suppo s he hypo hesis ha NADA is a b anch poin in
he syn hesis o he compa ible solu es ec oine and hyd oxyec-
oine is also p esen ed in his pape .
MATERIALS AND METHODS
S ains and g ow h condi ions. H. elonga a DSM 3043 (wild ype) (27),
CHR61 (a spon aneous i ampin- esis an mu an o he wild- ype s ain),
CHR62 (ec A::Tn1732), and CHR63 (ec C::Tn1732) ha e been desc ibed p e i-
ously (3, 6). H. elonga a s ains we e g own in de ined M63 medium con aining
20 mM glucose as he sole ca bon sou ce (7). The pH o he medium was
* Co esponding au ho . Mailing add ess: Depa amen o de Mic o-
biologı´a y Pa asi ologı´a, Facul ad de Fa macia, Uni e sidad de Se illa,
41012 Se ille, Spain. Phone: 34-954-556765. Fax: 34-954-628162.
E-mail: [email p o ec ed].
† P esen add ess: Depa amen o de Bio ecnologı´a Mic obiana,
Cen o Nacional de Bio ecnologı´a, CSIC, Can oblanco, 28049 Mad id,
Spain.
3774
on Ma ch 3, 2016 by INSTITUTO DE PARASITOLOGFA Yh p://aem.asm.o g/Downloaded om
adjus ed o 7.2 wi h KOH. Liquid cul u es we e incuba ed a 37°C in an o bi al
shake a 200 pm. Cell g ow h was moni o ed by measu ing he op ical densi y
a 600 nm (OD
600
).
To s udy he e ec o salini y on he accumula ion o o ganic solu es, cul u es
we e g own in media con aining 0.5 o 1.5 M NaCl un il hey eached he la e
exponen ial phase (OD
600
, 1.0 o 1.2). The e ec o he g ow h phase on he
accumula ion o solu es was examined by g owing cells in medium con aining 1.0
M NaCl. Fo he expe imen s pe o med wi h mu an CHR62 ha we e designed
o in es iga e he ec oine-hyd oxyec oine in e con e sion, media we e supple-
men ed wi h 1 mM NADA, 1 mM ec oine, o 1 mM hyd oxyec oine. Cells we e
ha es ed by cen i uga ion (8,000 ⫻g, 30°C, 10 min).
Cell p o ein con en s. The p o ein con en s o he cells we e de e mined by he
B ad o d assay (1) a e cell lysis wi h 1 M NaOH (100°C, 10 min) and neu al-
iza ion wi h 1 M HCl.
Ex ac ion and de e mina ion o in acellula solu es by NMR spec oscopy.
Cell pelle s ob ained om cul u es o H. elonga a we e ex ac ed wice wi h
boiling 80% e hanol by he me hod o Reed e al. (21), modi ied as p e iously
desc ibed by Ma ins and San os (17). F eeze-d ied ex ac s we e dissol ed in
D
2
O o nuclea magne ic esonance (NMR) analysis. Solu es we e quan i ied by
1
H NMR by using o ma e as an in e nal concen a ion s anda d. Fo quan i i-
ca ion pu poses, spec a we e acqui ed wi h a 6-␮s pulse wid h (co esponding o
a 60° lip angle) and a epe i ion delay o 65 s.
1
H NMR spec a we e eco ded
a 300.14 MHz wi h a B uke model AMX300 spec ome e equipped wi h a
5-mm-diame e b oad-band in e se p obe head.
13
C NMR spec a we e ob ained
wi h a B uke model DRX500 spec ome e a 125.77 MHz.
Pu i ica ion o NADA. An e hanol-soluble ex ac o an H. elonga a CHR63
cul u e g own in M63 medium supplemen ed wi h 0.75 M NaCl was applied on o
an ac i a ed Dowex 50W-X8 ca ion-exchange esin column (16 by 2.8 cm).
Elu ion was pe o med wi h a linea g adien o pe chlo ic acid (0 o 2 M) a a
low a e o 2.5 ml min
⫺1
. Aliquo s o each ac ion we e neu alized wi h KOH
and we e analyzed o he p esence o amino acid de i a i es by using he
ninhyd in es . F ac ions con aining amino acid de i a i es we e eeze-d ied and
analyzed by
1
H NMR spec oscopy. The ac ions ha elu ed be ween 1.5 and 1.7
M pe chlo ic acid con ained hyd oxyec oine and NADA, and he ac ions ha
elu ed be ween 1.7 and 2 M pe chlo ic acid con ained pu e NADA. Samples
con aining pu e NADA we e pooled, and sal s we e emo ed by passage h ough
an ion e a da ion column ( ype AG11 A8; 45 by 2.5 cm). A e lyophiliza ion,
ul apu e wa e was added o ob ain a 0.6 M NADA solu ion. This solu ion was
s o ed a ⫺20°C un il i was used. No ca ions (Na
⫹
o K
⫹
) we e de ec ed by
plasma emission spec oscopy pe o med wi h a Jobin Y on spec ome e (mod-
el JY24).
Pu i ica ion o glucosylglyce a e. An e hanol-soluble ex ac o an H. elonga a
CHR63 cul u e g own in M63 medium con aining 1.0 M NaCl was applied o an
ac i a ed Dowex 50W-X8 esin (H
⫹
o m) column and elu ed wi h dis illed
wa e . The elu ed ac ions we e analyzed o ca bohyd a e by he me hod o
Dubois e al. (9). The ac ions con aining ca bohyd a es we e pooled, neu al-
ized wi h 1 M KOH, lyophilized, and dissol ed in D
2
O; hese ac ions we e used
o
1
H NMR analysis.
Measu emen s o enzyme ac i i y. Rabbi muscle LDH, which was ob ained
om he supplie as a suspension in ammonium sul a e, was applied o a ype
PD-10 column and elu ed wi h 10 mM po assium phospha e bu e (pH 7.6).
LDH ac i i y was assayed by moni o ing oxida ion o NADH on he basis o
measu emen s o abso bance a 340 nm, as de e mined wi h a spec opho ome e
(model DW-2; Olis) equipped wi h a he mos a -equipped cu e e holde a
30°C. The s anda d eac ion mix u e con ained 80 mM T is-HCl (pH 7.6), 1.6
mM py u a e (sodium sal ), 0.2 mM NADH, and 0.25 ␮g o enzyme in a o al
olume o 1 ml (25). The eac ion was ini ia ed by adding py u a e.
The me hod p e iously desc ibed o de e mining p olyl hyd oxylase ac i i y
was used o de ec pu a i e NADA hyd oxylase ac i i y in c ude cell ex ac s o
H. elonga a CHR63 (24). To do his, cells we e g own in M63 medium supple-
men ed wi h 1.5 M NaCl and ha es ed du ing he exponen ial g ow h phase; he
cell pelle was suspended in a saline solu ion (1.5 M NaCl) con aining he
p o ease inhibi o phenylme hylsul onyl luo ide a a concen a ion o 1 mM, 1
mM EDTA, 1 mM MgCl
2
, and 2 mg o DNase ml
⫺1
. A e he cells we e b oken
by passage h ough a F ench p ess, he esul ing ex ac was cen i uged
(15,000 ⫻g, 30 min, 4°C) o emo e he cell deb is. The supe na an solu ion was
dialyzed o e nigh agains 20 mM phospha e bu e (pH 7.2).
The mal s abili y assays. The he mal s abili y assay mix u es con ained 50 ␮g
o LDH ml
⫺1
wi h o wi hou one o he ollowing solu es: KCl, DA, NADA,
ec oine, and hyd oxyec oine. All o he solu es we e used a a inal concen a ion
o 0.5 M. Mix u es we e placed in Eppendo ubes and incuba ed a 50 o 55°C
in a wa e ba h, and aliquo s we e wi hd awn a di e en imes. The aliquo s
we e cooled in an ice ba h and immedia ely assayed o enzyme ac i i y. Resul s
a e p esen ed below as pe cen ages o ac i i y compa ed wi h he ac i i ies o
aliquo s kep a oom empe a u e.
Chemicals. Type II abbi muscle LDH (E.C. 1.1.1.27) was ob ained om
Sigma. Dowex AG 50W-X8 and he ion e a da ion esin AG 11 A8 we e
pu chased om Bio-Rad Labo a o ies (Richmond, Cali .). The PD-10 column
was ob ained om Pha macia Fine Chemicals (Uppsala, Sweden). DA was
ob ained om Sigma. Ec oine and hyd oxyec oine we e gene ously p o ided by
Bi op GmbH.
RESULTS
Iden i ica ion o he o ganic solu es ha accumula ed in H.
elonga a mu an s ain CHR63. The
13
C NMR spec a o e h-
anol ex ac s o he mu an CHR63 con ained se e al se s o
esonances ha we e assigned o NADA, hyd oxyec oine, ec-
oine, and glucosylglyce a e (Fig. 2). Small amoun s o glu a-
ma e, lac a e, ehalose, and alanine we e de ec ed in he
1
H
NMR spec a o he same ex ac s (da a no shown). Reso-
nances we e assigned by compa ison wi h p e iously published
chemical shi alues (6, 12, 23, 29). In mos cases assignmen s
we e con i med by spiking samples wi h he pu e compounds.
The only excep ion was glucosylglyce a e; assignmen o his
o ganic solu e was con i med by pa ially pu i ying an e hanol
ex ac and pe o ming
1
H and
13
C NMR analyses. The
13
C
NMR spec um con ained only nine majo ca bon esonances
(a 61.0, 63.6, 70.0, 72.0, 72.7, 73.7, 79.3, 97.9, and 177.5 ppm),
whose chemical shi s ag eed wi h he chemical shi s p e i-
ously ob ained o his compound in Me hanohalophilus sp. and
he blue-g een alga Agmenellum quad uplica um (14, 22).
E ec o NADA as an enzyme he mos abilize . Since i had
been shown ha NADA could unc ion as a compa ible solu e
in H. elonga a (6), we decided o in es iga e whe he NADA
could also ac as an enzyme s abilize . This compound was
pu i ied om mu an CHR63. The
1
H NMR spec um o he
pu e compound is shown in Fig. 3. The abili y o NADA o
s abilize abbi muscle LDH was compa ed wi h he abili ies o
ec oine, hyd oxyec oine, and DA, he immedia e p ecu so o
NADA in membe s o he genus Halomonas (Fig. 4A). A
50°C, NADA had a signi ican he mos abilizing e ec on
LDH; his e ec was su passed only by he e ec o hyd oxyec-
oine. Howe e , when he enzyme was incuba ed a a highe
empe a u e, 55°C, hyd oxyec oine was he only solu e ha
signi ican ly p o ec ed he enzyme agains he mal inac i a ion,
and NADA was a poo s abilize (Fig. 4B). The po assium sal
o DA had a weak s abilizing e ec a 50°C, compa able o he
e ec o KCl.
FIG. 1. Ec oine biosyn he ic pa hway. See e e ence 19. The genes o he h ee enzymes in ol ed in ec oine syn hesis, DA ace yl ans e ase, L-DA ansaminase,
and ec oine syn hase, a e designa ed ec A,ec B, and ec C, espec i ely (3). CoA, coenzyme A.
VOL. 65, 1999 ROLE OF NADA IN ENZYME STABILIZATION 3775
on Ma ch 3, 2016 by INSTITUTO DE PARASITOLOGFA Yh p://aem.asm.o g/Downloaded om
Accumula ion o o ganic solu es by mu an CHR63 as a
unc ion o salini y and g ow h phase. In addi ion o he majo
compound NADA, which was p e iously de ec ed in mu an
CHR63, we iden i ied ec oine, hyd oxyec oine, and glucosylg-
lyce a e as signi ican membe s o he in acellula pool o
solu es in his o ganism. We in es iga ed he accumula ion o
solu es as a unc ion o he sal concen a ion in he medium
and he g ow h phase (Fig. 5). NADA was by a he p edom-
FIG. 3.
1
H NMR (300-MHz) spec um o NADA a e pu i ica ion om H. elonga a CHR63.
FIG. 2.
13
C NMR spec um o he c ude ex ac o H. elonga a mu an s ain CHR63 g own in M63 medium supplemen ed wi h 1.5 M NaCl. The majo solu es we e
NADA, hyd oxyec oine (H), ec oine (E), glu ama e (G), and glucosylglyce a e (GG). Fo ma e (F) was added o he sample as an in e nal concen a ion s anda d.
3776 CA
´NOVAS ET AL. APPL.ENVIRON.MICROBIOL.
on Ma ch 3, 2016 by INSTITUTO DE PARASITOLOGFA Yh p://aem.asm.o g/Downloaded om
inan solu e and accumula ed a all o he salini ies and g ow h
s ages examined. H. elonga a CHR63 g ows op imally in he
p esence o NaCl concen a ions anging om 0.75 o 1.0 M
(6). The concen a ion o he o al pool o o ganic solu es was
main ained a app oxima ely 2.8 ␮mol mg o p o ein
⫺1
a NaCl
concen a ions up o 0.75 M. A he highes NaCl concen a-
ion used (1.5 M), he concen a ion o he o al solu e pool
inc eased o 7.2 ␮mol mg o p o ein
⫺1
, and NADA accoun ed
o 80% o he o al pool. Howe e , hyd oxyec oine (12%) and
ec oine (6%) also made ele an con ibu ions. A NaCl con-
cen a ions o 0.75 o 1.5 M, he le els o NADA, ec oine, and
hyd oxyec oine inc eased 2.4-, 4.7-, and 10- old, espec i ely,
showing ha accumula ion o hese solu es is osmo egula ed in
s ain CHR63. In con as , glucosylglyce a e did no unc ion
as a compa ible solu e, since i accumula ed p e e en ially a
low le els o salini y. Low le els o glu ama e, ehalose, ala-
nine, and lac a e we e also de ec ed, bu hey we e no accu-
a ely quan i ied; he combined con en s o hese mino solu es
did no exceed 10% o he o al solu e pool (Fig. 5A).
The le els o solu es we e clea ly a ec ed by he g ow h
phase (Fig. 5B). The e we e no di e ences in he concen a-
ions o he cy oplasmic pools o compounds in he ea ly
(OD
600
, 0.5) and la e (OD
600
, 1) exponen ial phases o g ow h.
Howe e , du ing he s a iona y phase (OD
600
, 1.6 o 1.8), he
concen a ions o all solu es inc eased conside ably. I is no e-
wo hy ha a h ee old inc ease in he le els o hyd oxyec oine
was de ec ed du ing he s a iona y phase o g ow h (Fig. 5B).
E idence ha NADA is a b anch poin o he syn hesis o
ec oine and hyd oxyec oine. The obse a ion ha in H. elon-
ga a mu an CHR63 he hyd oxyec oine/ec oine a io was con-
side ably highe han he a io in he wild- ype s ain (6; his
s udy) led us o he hypo hesis ha NADA could be a di ec
p ecu so o he syn hesis o hyd oxyec oine ia an al e na i e
pa hway di e en om he pa hway p e iously p oposed (in
which ec oine is used as a p ecu so ) (6, 13). In o de o es
his hypo hesis, wo mu an s ains, CHR62 (ec A::Tn1732)
and CHR63 (ec C::Tn1732), and he wild- ype s ain o H.
elonga a we e cul i a ed in M63 medium con aining 0.65 M
NaCl, ha es ed a an OD
600
o 0.9, and examined o he
p esence o ec oine, hyd oxyec oine, and NADA. Mu an
CHR62, which is a ec ed in he DA ace yl ans e ase gene
(ec A), did no syn hesize ec oine o hyd oxyec oine (Fig. 6),
whe eas mu an CHR63, which is a ec ed in he ec oine syn-
hase gene (ec C), and he wild- ype s ain syn hesized hy-
d oxyec oine and ec oine wi h ela i e p opo ions o 1.3:1 and
0.4:1, espec i ely.
To assess he occu ence o di ec in e con e sion be ween
hyd oxyec oine and ec oine, mu an CHR62 was g own in M63
medium supplemen ed wi h 1 mM NADA, 1 mM ec oine, o 1
mM hyd oxyec oine, and 2.0 M NaCl. Hyd oxyec oine and
ec oine a a mola a io o 2:1 we e syn hesized om NADA.
When ec oine was p o ided, he hyd oxyec oine/ec oine a io
was 0.7, and his a io inc eased o 7.0 when hyd oxyec oine
was p o ided ins ead o ec oine ( esul s no shown). These
esul s show no only ha hyd oxyec oine can be syn hesized
FIG. 4. E ec s o NADA (■), ec oine (Ec ) (Œ), hyd oxyec oine (Hec ) (E),
po assium DA (✳), and po assium chlo ide (}) on he mal inac i a ion o abbi
muscle LDH. (A) The enzyme was incuba ed a 50°C in he absence (F)o in he
p esence o one o he solu es, and samples we e wi hd awn a di e en imes.
(B) The enzyme was incuba ed a 50 o 55°C o 10 min in he p esence o in he
absence o one o he solu es. The ac i i y o he enzyme was immedia ely
assayed. Values a e exp essed as pe cen ages o ac i i y compa ed o he ac i i y
o he enzyme incuba ed a oom empe a u e. All o he esul s a e a e ages o
he alues om a leas wo independen expe imen s. The s anda d de ia ion
was equal o o less han 10%.
FIG. 5. Accumula ion o solu es in H. elonga a CHR63. (A) Cells we e g own
in M63 medium supplemen ed wi h di e en sal concen a ions and samples
we e wi hd awn in he la e exponen ial phase. (B) Cells we e g own in M63
medium supplemen ed wi h 1.0 M NaCl, and samples we e aken a di e en
g ow h s ages. Tu bidi y (OD
600
) alues o 0.5 and 1.0 co espond o he ea ly
and la e exponen ial phases o g ow h, espec i ely, and alues o 1.6 and 1.8
co espond o he s a iona y phase. The solu es we e iden i ied and quan i ied by
1
H NMR by using e hanol cell ex ac s. The alues a e he means o alues om
a leas wo independen de e mina ions, and he indi idual alues did no di e
by mo e han 20%.
VOL. 65, 1999 ROLE OF NADA IN ENZYME STABILIZATION 3777
on Ma ch 3, 2016 by INSTITUTO DE PARASITOLOGFA Yh p://aem.asm.o g/Downloaded om
di ec ly om ec oine bu also ha con e sion o ec oine o
hyd oxyec oine is a e e sible eac ion, which unde he con-
di ions es ed seemed o be a o ed in he di ec ion o hy-
d oxyec oine syn hesis.
In summa y, in he o ganisms in which he in acellula
concen a ion o NADA was high, he hyd oxyec oine le el
was signi ican ly highe han he le el o ec oine, sugges ing
ha in H. elonga a DSM 3043 NADA is he b anch poin in he
biosyn he ic pa hway o ec oines (Fig. 6).
DISCUSSION
The s abilizing e ec s o osmoly es, such as glyce ol and
suga s, on enzymes and whole cells we e well-known long be-
o e he e m compa ible solu e was in oduced by B own in
1976 (2). Mo e ecen ly, compa ible solu es ha e ecei ed con-
side able a en ion due o hei ema kable p o ec ion o en-
zymes agains hea , sal , eezing, and d ying. Al hough gene al
mechanisms o his p o ec ion ha e been pos ula ed, he de-
g ee o s abiliza ion achie ed seems o depend on he speci ic
enzyme-solu e pai unde in es iga ion (20; see e e ences 8
and 10 o e iews).
The labile enzyme abbi muscle LDH has o en been used
as a model sys em o enzyme s abiliza ion es s (10, 15, 20).
This enzyme is e y sensi i e o high empe a u es and also is
apidly inac i a ed by eeze- haw ea men (10). We ound
ha NADA, he p ecu so o ec oine, p o ec ed his enzyme
agains inac i a ion by hea a 50°C. This p o ec ion was com-
pa able o ha o hyd oxyec oine and much be e han ha o
ec oine o DA. This was su p ising, since NADA can be con-
side ed he hyd olyzed linea o m o ec oine.
To ou knowledge, he s abiliza ion p ope ies o solu es
belonging o he N-ace yla ed diamino acid g oup (such as
N␦-ace ylo ni hine and Nε-ace yllysine) ha e been s udied by
using eeze- haw cycles, bu no he mal p o ec ion esul s
ha e been epo ed. An impo an ea u e o his kind o com-
pound is ha he ace yl moie y mus be a ached o he N-
e minal posi ion ␻ o p o ide comple e p o ec ion, and
␣-ace yla ed isome s a e poo e p o ec o s (6, 10). Al hough
NADA e y e icien ly p o ec ed agains LDH inac i a ion a
50°C, i did no ha e any s abilizing e ec a 55°C. In con as ,
solu es, such as mannosylglyce a e, which a e accumula ed by
he mophilic and hype he mophilic o ganisms, and hy-
d oxyec oine a e able o p o ide ema kable p o ec ion agains
inac i a ion a high empe a u es (10, 20).
I was no known un il e y ecen ly ha NADA plays a ole
FIG. 6.
1
H NMR (300-MHz) spec a o e hanol ex ac s o H. elonga a sal -sensi i e mu an s ains CHR62 (ec A::Tn1732) and CHR63 (ec C::Tn1732) and o
CHR61 (wild- ype s ain) g own in M63 medium supplemen ed wi h 0.65 M NaCl. The a ows indica e he posi ion o he hyd oxyec oine esonance. Abb e ia ions:
H, hyd oxyec oine; E, ec oine. The biosyn he ic pa hway o ec oines om DA is also shown. The solid and dashed lines indica e es ablished and p oposed s eps,
espec i ely. The le e X ep esen s an in e media e in hyd oxyec oine biosyn hesis ha has no been iden i ied so a .
3778 CA
´NOVAS ET AL. APPL.ENVIRON.MICROBIOL.
on Ma ch 3, 2016 by INSTITUTO DE PARASITOLOGFA Yh p://aem.asm.o g/Downloaded om

in he osmo ic adap a ion o bac e ia (6); he e we show ha
his compound can also be added o he epe o y o enzyme
s abilize s. Howe e , u he s udies on he s abilizing e ec o
NADA on a a ie y o enzymes subjec ed o di e en ypes o
s ess mus be pe o med in o de o e alua e he abili y o
NADA as a gene al enzyme s abilize . In his espec , H. elon-
ga a sal -sensi i e mu an CHR63 has g ea po en ial in bio-
echnology as a sou ce o a cock ail o compa ible solu es ha
comp ehend p ima ily NADA and hyd oxyec oine, wo e y
e icien enzyme s abilize s.
The da a o accumula ion o in acellula solu es as a unc-
ion o he NaCl concen a ion in he medium s ongly sugges
ha syn hesis o NADA, ec oine, and hyd oxyec oine is osmo-
egula ed in mu an CHR63. Howe e , his o ganism is no
able o g ow in he p esence o NaCl concen a ions g ea e
han 1.5 M (6), in con as o he wild- ype s ain. This sugges s
ha ec oines a e p obably mo e e icien osmoly es in i o han
NADA.
Al hough hyd oxyec oine undoub edly can be used as an
enzyme-s abilizing agen (10), he biosyn he ic pa hway o his
compound has no been de e mined. The esul s o p e ious
s udies pe o med wi h H. elonga a mu an s impai ed in he
syn hesis o ec oine sugges ed ha hyd oxyec oine migh be
syn hesized di ec ly by ec oine hyd oxyla ion (6, 13). In ac ,
in e con e sion o ec oine and hyd oxyec oine was demon-
s a ed by Go¨lle e al. (13) wi h mu an SAA4, which con ains
a mu a ion in he same gene as H. elonga a CHR62. These
esul s we e con i med in he p esen wo k. In addi ion, we
ob ained e idence ha he e is an al e na i e ou e, in which
NADA is con e ed in o hyd oxyec oine wi hou he in ol e-
men o ec oine as an in e media e me aboli e. In he pa hway
p oposed he e (Fig. 6), hyd oxyec oine is syn hesized ia a
wo-s ep pa hway in ol ing hyd oxyla ion o NADA o p o-
duce 3-hyd oxyl-N␥-ace yldiaminobu y a e, which is subse-
quen ly con e ed o hyd oxyec oine by he ac ion o a pu a i e
hyd oxyec oine syn hase. We looked o his ac i i y in ex ac s
o H. elonga a CHR63 cells, bu we ailed o de ec i , p obably
due o ins abili y o he enzyme.
The obse a ion ha hyd oxyec oine p edomina es o e ec-
oine in o ganisms wi h high in acellula concen a ions o
NADA (mu an s CHR63 and CHR62 g own in he p esence o
NADA) sugges s ha he i s enzyme in ol ed in he al e na-
i e pa hway o syn hesis o hyd oxyec oine has a lowe a ini y
o he subs a e han ec oine syn hase has. Howe e , he el-
a i e con ibu ions o he wo pa hways o syn hesis o hy-
d oxyec oine in he wild- ype s ain o H. elonga a canno be
deduced om he da a p esen ed he e. Wo k is in p og ess o
ully elucida e he pa hways o biosyn hesis o hyd oxyec oine
by isola ing he genes and cha ac e izing he key enzymes.
ACKNOWLEDGMENTS
We hank Bi op GmbH o kindly p o iding ec oine and hyd oxyec-
oine.
D. Ca´no as and N. Bo ges con ibu ed equally o his wo k.
D. Ca´no as acknowledges a ellowship om he Spanish Minis e io
de Educacio´n y Ciencia. This wo k was suppo ed by he Eu opean
Commission BIOTECH P og amme Ex emophiles as Cell Fac o ies
(g an BIO4-CT96-0488), by PRAXIS XXI and FEDER, Po ugal
(g an PRAXIS/2/2.1/BIO/1109/95), by he Spanish Minis e io de Edu-
cacio´n y Cul u a (g an s PB97-0722 and BIO97-1876-CE), and by he
Jun a de Andalucı´a.
REFERENCES
1. B ad o d, M. M. 1976. A apid and sensi i e me hod o he quan i a ion o
mic og am quan i ies o p o ein u ilizing he p inciple o p o ein-dye bind-
ing. Anal. Biochem. 72:248–254.
2. B own, A. D. 1976. Mic obial wa e s ess. Bac e iol. Re . 40:803–846.
3. Ca´no as, D., C. Va gas, M. I. Calde o´n, A. Ven osa, and J. J. Nie o. 1998.
Cha ac e iza ion o he genes o he biosyn hesis o he compa ible solu e
ec oine in he mode a ely halophilic bac e ium Halomonas elonga a DSM
3043. Sys . Appl. Mic obiol. 21:487–497.
4. Ca´no as, D., C. Va gas, L. N. Csonka, A. Ven osa, and J. J. Nie o. 1996.
Osmop o ec an s in Halomonas elonga a: high-a ini y be aine anspo sys-
em and choline-be aine pa hway. J. Bac e iol. 178:7221–7226.
5. Ca´no as, D., C. Va gas, L. N. Csonka, A. Ven osa, and J. J. Nie o. 1998.
Syn hesis o glycine be aine om exogenous choline in he mode a ely halo-
philic bac e ium Halomonas elonga a. Appl. En i on. Mic obiol. 64:4095–
4097.
6. Ca´no as, D., C. Va gas, F. Iglesias-Gue a, L. N. Csonka, D. Rhodes, A.
Ven osa, and J. J. Nie o. 1997. Isola ion and cha ac e iza ion o sal -sensi i e
mu an s o he mode a e halophile Halomonas elonga a and cloning o he
ec oine syn hesis genes. J. Biol. Chem. 272:25794–25801.
7. Cohen, G. N., and R. H. Rickenbe g. 1956. Concen a ion speci ique e e s-
ible des aminoacides chez E. coli. Ann. Ins . Pas eu (Pa is) 91:693–720.
8. da Cos a, M. S., H. San os, and E. A. Galinski. 1998. An o e iew o he ole
and di e si y o compa ible solu es in Bac e ia and A chaea. Ad . Biochem.
Engl. Bio echnol. 61:117–153.
9. Dubois, M., K. A. Gilles, J. K. Hamil on, P. A. Rebe s, and F. Smi h. 1956.
Colo ime ic me hod o de e mina ion o suga s and ela ed subs ances.
Anal. Chem. 28:350–356.
10. Galinski, E. A. 1993. Compa ible solu es o halophilic eubac e ia: molecula
p inciples, wa e -solu e in e ac ion, s ess p o ec ion. Expe ien ia 49:487–
496.
11. Galinski, E. A. 1995. Osmoadap a ion in bac e ia, p. 273–328. In R. K. Poole
(ed.), Ad ances in mic obial physiology. Academic P ess, London, Uni ed
Kingdom.
12. Galinski, E. A., H. P. P ei e , and H. G. T u¨pe . 1985. 1,4,5,6-Te ahyd o-
2-me hyl-4-py imidineca boxylic acid. A no el cyclic amino acid om halo-
philic pho o ophic bac e ia o he genus Ec o hio hodospi a. Eu . J. Bio-
chem. 149:135–139.
13. Go¨lle , K., A. O e , and E. A. Galinski. 1998. Cons uc ion and cha ac e -
iza ion o an NaCl-sensi i e mu an o Halomonas elonga a impai ed in
ec oine biosyn hesis. FEMS Mic obiol. Le . 161:293–300.
14. Kollman, V. H., J. L. Hanne , R. E. London, E. G. Adame, and T. E. Walke .
1979. Pho osyn he ic p epa a ion and cha ac e iza ion o
13
C-labeled ca bo-
hyd a es in Agmenellum quad uplica um. Ca bohyd . Res. 73:193–202.
15. Lamosa, P., A. Bu ke, R. Peis , R. Hube , M. Y. Liu, G. Sil a, C. Rod igues-
Pousada, J. LeGall, C. Maycock, and H. San os. Unpublished da a.
16. Liss, I. 1962. N-Ace yldiamniobu e sa¨u e, eine neue Aminosa¨u e aus dem
La ex on Eupho bia pulche ima Willd ex Klo zsch. Phy ochemis y 1:87–88.
17. Ma ins, L. O., and H. San os. 1995. Accumula ion o mannosylglyce a e
and di-myo-inosi ol-phospha e by Py ococcus u iosus in esponse o salini y
and empe a u e. Appl. En i on. Mic obiol. 61:3299–3303.
18. Ono, H., K. Sawada, N. Khunajak , T. Tao, M. Yamamo o, M. Hi amo o, A.
Shinmyo, M. Takano, and Y. Mu ooka. 1999. Cha ac e iza ion o biosyn-
he ic enzymes o ec oine as a compa ible solu e in a mode a ely halophilic
eubac e ium, Halomonas elonga a. J. Bac e iol. 181:91–99.
19. Pe e s, P., E. A. Galinski, and H. G. T u¨pe . 1990. The biosyn hesis o
ec oine. FEMS Mic obiol. Le . 71:157–162.
20. Ramos, A., N. D. H. Ra en, R. J. Sha p, S. Ba olucci, M. Rossi, R. Cannio,
J. Lebbink, J. Van de Oos , W. M. de Vos, and H. San os. 1997. S abiliza ion
o enzymes agains he mal s ess and eeze-d ying by mannosylglyce a e.
Appl. En i on. Mic obiol. 63:4020–4025.
21. Reed, R. H., L. J. Bo owi zka, M. A. Mackay, J. A. Chudek, R. Fos e , S. R. C.
Wa , D. J. Moo e, and W. D. P. S ewa . 1984. O ganic solu e accumula ion
and osmo ic s ess in cyanobac e ia. J. Gen. Mic obiol. 130:1–4.
22. Robe son, D. E., M. C. Lai, R. P. Gunsalus, and M. F. Robe s. 1992.
Composi ion, a ia ion, and dynamics o majo osmo ic solu es in Me hano-
halophilus s ain FDF1. Appl. En i on. Mic obiol. 58:2438–2443.
23. Se e in, J., A. Wohl a h, and E. A. Galinski. 1992. The p edominan ole o
ecen ly disco e ed e ahyd opy imines o osmoadap a ion o halophilic
eubac e ia. J. Gen. Mic obiol. 138:1629–1638.
24. Tude man, L., R. Myllyla¨, and K. I. Kil i ikko. 1977. Mechanism o he
p olyl hyd oxylase eac ion. Eu . J. Biochem. 80:341–348.
25. Vassaul , A. 1987. Lac a e dehyd ogenase. UV-me hod wi h py u a e and
NADH, p. 118–126. In H. U. Be gmeye (ed.), Me hods o enzyma ic anal-
ysis, 3 d ed., ol. III. VCH, Weinheim, Ge many.
26. Ven osa, A., J. J. Nie o, and A. O en. 1998. Biology o mode a ely halophilic
ae obic bac e ia. Mic obiol. Mol. Biol. Re . 62:504–544.
27. V eeland, R. H., C. D. Li ch ield, E. L. Ma in, and E. Ellio . 1980. Halomo-
nas elonga a, a new genus and species o ex emely sal - ole an bac e ia. In .
J. Sys . Bac e iol. 30:485–495.
28. V eeland, R. H., and E. L. Ma in. 1980. G ow h cha ac e is ics, e ec s o
empe a u e, and ion speci ici y o he halo ole an bac e ium Halomonas
elonga a. Can. J. Mic obiol. 26:746–752.
29. Wohl a h, A., J. Se e in, and E. A. Galinski. 1990. The spec um o com-
pa ible solu es in he e o ophic eubac e ia o he amily Halomonadaceae.
J. Gen. Mic obiol. 136:705–712.
VOL. 65, 1999 ROLE OF NADA IN ENZYME STABILIZATION 3779
on Ma ch 3, 2016 by INSTITUTO DE PARASITOLOGFA Yh p://aem.asm.o g/Downloaded om