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Gene
Cloning And ExpressionOf 3-Ilydroxybutyrate D€hydrogenase
sp.
From Locally IsolatedPseudomonas
N.NI. Nik Azmi, Rosli Mohd. Itlias, S.M. SyedAnuar Faua'ad,
H.D. Hrbib Abdullah Fikri, S.WanNor Azlinda, S' Y' Lau
Depa nentofEioprccess EngineeiiS FKKKSA Unitersiti Tekhologi Malovsia'
Johor
81310UTM Sk
'tai
Abstract
The 3-HBDH gete from Prtldauorus sP. was
amplifi€d. cloned, and sequenced The deduc.d
r m ino a" i d ,e q u e n c e q J s h i Sh l y m a l c h eddi l h
in DDBi/CenBanUEMBL.Bui
3-HBDH sequences
Pseudonanas sp. 3 HBDH was lsck of the
C-terbinal region found in mammalianenzymes
c onr r nr nga .i p rdb ;rd i i 8 J o ma .nIh rr i \ i m por' rnt
f or r he t H a D H " .c rr\i l ) l h e l ' d ' \Ll B l L.
cellstrMstomed with the resultantplasmid,PBDH'
I s hos ed J .H BD H ' J c ti v i l y . T h e n u .l eorrde
s eqJ enc e o f re .o m b i n a n l p B D H -l Ind(are
functionaloligomerscomposedof subunitsof 255
amino acid with a calculatedM, of 26,855Da and
pro\ed on S D S P A C F. A mmon;um " Jl ;te
re' ul redi n l ow )i el d ol thee /)mc bul
fmcl ;onal ;on
its aclivity was compambly high rvith othel
3-HBDHS. The recombiranl enzyme showed a
broadrangeof ' r.b:l i (y \i rh re.pe.l o D H and
Introduction
TI H B D H)
J-l )d o!)bLr)rare del ' )dogen-e
(EC 1.I.I.30) catalyzesthe revesible oxidationof
rre, uti l i zi ngni coun e
Io acel oare
hydro{ybur}rare
dr a .oenzymel l l !s \hoqn i n
dJeni ne
d;nucl eol i de
Figurc la.
!
iRl-3.llydrorvburanoate: NAo'oxidor€du.tasaiEC l. 1. 1 301'
..3 1lya.Dx,/but?rnroNAO- ,
: Ac€r.ac6trlc ' NADrr i ll_
Fisute 1a - Reve.sible rcdctian mechanisn aJ 3'HBDH.
The substrute,3-hldtorbut!rcte acid was otidizeLl to acetoacetate
Thk reactionprovidesa basisfor detemination.t
and bers_h)d'oxyburvric
keronebod;es,.cetoacel.re
acid.Deficien!insulinavailabilityin diabetescauses
decreaseof glucoseintake and increasein ketone
b" die. r o md ' i o n a n d m a y .a J s e dtdb€l ;c
keroacidosis(DKA) [2]. To prevent this. the
of ketonebodis in blood or urin€ can
concentration
be monitored with williamson method t3l
diazoniumsalt method[4] and the latestketo'fiim
method t5l using 3-HBDH as part of lhe
detemination procedures.For a long tim€, sequence
informstion was limited to mamnalian 3-HBDHS
such as human heart [6], bovine heart I7l and mt
liver mitochondriaalthough3-HBDHS from a
number of bacterial sources had been partially
purified such as Rhodopseudononai sphetoi.ts lll
analvsl
andPotu.o..^ Jen nfi.anrllgl. Sequence
revealedlhal mammalian3-HBDH is found belongs
of
superf.mily
ro
rhe
'holl_chain
which consists of
dehydrogenasevreductases,
coenzymebinding followedby the catalyricdomain
at N-terminai.In this study,we presentthe cloning
and sequencing of 3-HBDH isolated from
Pseudomonassp.laczlly isolat€d from soil, as well
262
as panial purincation and characterization of fte
recombinantproleiD expressedin E coli
Materials And Methods
Pseudomona.ssp. was isolated from soil and
storedin a glycerinstock G80C).E cali XLlblue was used as host , pGEMTeasy, and
pKl<223-2 wete used as vectors. Large scale
cutivation was cMied out in Nulrient broth
(1% meatextract,l% polypepton€,
0.5% NaCl)
DNA wasextmcted
sp.Chrc(nlJsomal
Pseudononas
by a sundard phenol €xtraction method. The
wercdesignedon ihe
oligoneucleotides
degenerated
basisof conservedaminoacid s€quenceamongthe
reported baclErial 3-HBDHS. The pnmers were
combin€d and by trial and enor, chaging the
annealingtemperatureof fte PCR condilion.PCR
wascarriedout in 50 gl reactionmixlurecontaining
GC buff€r II (Takara).PCR produclswere cloned
into pGEM-Teasy v€.tor system (Promega).
by
Posiliv€cloneswereusedfor DNA sequencing
SangermeihodusingAlFExpressDNA sequer'cer'
ISBN:983-2643-15-5
Pnt.edh!:
Rec(Inbiirrr sequencetrus comparcdNith odref3,
HB DLI Sus ir g B l .s t p fo g tu m(N C B I) v i .r i n teLne!
coupLedwirh Ceneryx-macsothvafe.E .rll XLI
blue ha$orlng pKK/BDH was lrerobicrllycultured
in 20lj! r esof N b ro thc o n ra i n i nagrrp i c i l l i na t 3?' C
for 17 hours. using r jff femrertor. Annotrium
Ithle fractionationrnd DEAE TOYOperrl colunn
\!cfe used i partial purification ol 3 HADH
r ec om binr r ta s i .d i c a re i r T rb l e l . A fre r .acn
purificrtion sreps. tracrions identificadon ol
I HBDH presenls,fonn ion ol difomazin rnerhod
eons is t of
s re l c ri o r n ri x l u re(1 .0 ml ) o i 1 0 0 mN I
T r is - HCl( pH 8 .5 ).! 2 ta T ri to nX-1 0 0 ,2 5 n D L
I h\drox\bLrrrric Na sl}lr. 2 mN'l NAD+, 0.001%
PhenrzineNIedro$rlfate(PIUS)1,0.017. nitroblue
tetr;lzolium salt (NBT) and 0.1 lnl oi enzyme.
Themrl and pH slability wefe tesiedon pa,ljalu
pudtlcd enzynrewith tenperatureineubrrionrarge
be$veen0-60C andpH fangeof6 to 9.5.
TdbleI .Pa iatpuriTcationstepsoJrccanbrntn
PBDH-1.
ResultsAnd Discussion
P CR pm duc .$ i rh e \p c .rc de z e o . 0 7 7 k o p
"hi ch
has a EcoRl and HindIII site at each foNard and
reverseprimer w6 produced.The PCR condition
c on. i. ledoi I ()c l e o f d e n J tu rl tro n
fo r 5 m .n dL
94oC.27 cycles of de.aluralion ar 95'C fo. 45 s,
annealingar 62oCfor45 s andextens;ona! 7lC for
I min. The reactjonfor primer extensioncontinued
l
.l Lrt.nxriondl Cot.rne
On Cherti..t an.|Lintu,.4t En!ir"\\.it)!
27\ tqr A\an 2OOJ, Unik:ti Matllstu Slruh K.td KirlLthl
ibf ? nin. The PCR product rveie clone \!rrh
pcBlvlTeasy vector rnd posirive .lones were
sequenced.
The open readingffame is found to oe
765 rucleorides and encoding 255 amino rcid
,fi gurc o/tu\.Jues$i rhr.rhrl areJ m.run,e..i /e
of 26,855 Da. Fasta sexrch of the recombinanr
enzr-,mergainsr tle GenBrnk databaseyieldcd
.,l no\r e\-1" s.\pl ) Inen)b!r\ of rre l ,o.r.ch-n
dehtdrogenase
supedh.rily (SCAD). Pru,.lon,rar
tetueinost 3-HBDtl sequenceh$ . vefy high
sinilarnies ot' 7770 in nuclcotidea d comparble
similarilies of 12Eo tn amino acid. One of tbe
conseA,ed domain G|XXXGTXGT? rt the
Nrermjnal is know lo be a NADH,h,indingsire lbr
nost SCAD. The catalyric center fe.ruring rhc
putaljveactilc site residuesScr, Tyr, and Lys afc
also prcsent.Ho$evef, the recombinrntcnzyme*
roI precededby a leaderpeptide nor iollowed by
\eql erce
l onr.;i i ng
I pi o Lr.d ,g
il
LPGAISDMIYIR which occursin most mamnrxli!r
lype 3-HBDitr [l5]. This suggens rl[i rlE
recombinan!enzymeis indcpendenton iipid for its
activity. Nevertheless.
mammal typc 3 HBDH and
the recombinantenzymehavequite similarfearrrcs.
Forexpressionin E.coli,the genewascloned;nroan
expression v@tor, pKK223 3 fexruring a rac
promoter.Followinga largescalecultivarionat 37 C,
overnrght.lhe cells were firsl cell disrupr lvrtx
Dynomill before applied into puiiication sreps
rT.bl e 2r.Tl e sfeci [c a.I\i ,) .c 55 L,rngw t rt .
h,gh comparablro
e Jl l rypesoi , H B D H \ ro d.re
The S D S .P qU Ernai y!s ol rhe en/yme.\otreJ
band of apprcximately26 kDa (Figure 2) but rbe
bandbecameunclearafter further pufificationwirh
ammoolum sulfale. The partially puritied
recombinant
enzymewas stableup to 40oCand 507,
activity was maintainedevenafter $e incubationar
55'C tor 15 mins (Figure 3). Thk is:t conrnst to
one $ld'type 3-HBDH from Rhodopreu.lononas
sphercids.wnich lost 70% of acriyity rapidty at
37t. 'l'heenzymewas stablebelweenpH 7 and ptt
9 and naximum acdvity was shown at pH 8,8.5
(FisL,rc3).
-------.-.--!'TI;KGKTI|'J
MGI,PPPPGRI'SRI,PGKU,S.TCDRINGERRILiLCST€FIPIGRRTYASAAIP-VESKAVI,59
r] glc-STGIC'GIA"ALAAOGMIVI]NGFCDAXT:EIqNGLT.AQHC\,IIVLYDEADI]S]iG-EA
IO V'IGSTS€ICLGIA'iSLIEACAIITLI,TICFCT1IDIA!_AQV.RA.IOVRAISIIPII)LSDV-AQ
60 vTccDsclctsLA&grJssKGFrJ-vFAccr't{liDttgIlDo4aLDstNgDRr,RTVQtNVFRSEE
9
6?
58
120
!OEGMiMDC.CWVAO
P.A€tuginoEe
HrlJlla h€a!!
240 DQ1,'RCAAI,INI{DGGI4VAQ
301 DAVTIIAIiTATTPIITRhIPUDYYT$'I]XXOII{TULPCA
I SD.i1I YIR
246
34{
Figure Ib - Alisn'nent oj qBDH-1 l'ith Pseudononas aeruginos.3-HBDE and Hunan h.art 3-HBDH. The
GXXXCXG notif and alsa lipid biadins sile in nlan'nalian 3-HBDH are shown in rcd antl green respecnveLy.
ISBN:983-26a3-15-5
263
Pn,cp?dd$ al hrflatiatal C.rlir.n.e On Chenicattrd Biortoc($ F'htinecars
Unirtsiti MaLd)siaSdbthK.t.Kn bntu
Arrd2fi1,
2/)-2yt
F Nut e2 SD SP AGE a n tl J s | o ItE Ptttn l bP ui fi e' l rccanbi l nnt3' H B D H l nneIreconl bi hant(ttude
t . " i 2 .,^ " t,,, t' ti ,' .1 ' tt(tt;F 3 H B D H (6TkD abal l di sB S A thdtsti bi ti ' ?stha' n1k1?l ' t51' td3e )
" . u, i, 5.
"t
I
Fi! , / e 3 ' Th . t M l
n n d p H s t d b ) l l t tp a t l e n \ '
Conclusion
lhe 3- HB D Hg e n ew a s\u c c c * i L l l y c mp l i fi edan' l
expressedby using pKK 223-3 vector. The high
an
stabilily of lhe reconrbiqnl enzvme
's
p
h
e
n
omenon
T
h
e
d
i
a
g
n
o
"
i
c
f
o
r
c
l
i
n
i
c
d
l
dd\ ant age
of enzyme denatuntion after ammonium sulfate
n rh en e a rf-rure
nr ec ipr l. r i ocn0 n b e i n te c ri a a tei d
purification
meihod,
a reasonable
With an efficient
amount of p re enzyme can be obtained The
stnictureand functions are to be clarified in tbe
Aeklowledgem€nt
We !!ish 10thankUniaer$ti TeknologiMalaysia
for grantingus financiaLsupp€'rvia short'term
researchgrantvote 72129.
264
Reterences
tll
t2l
LJI
Pl
t5l
Bersmeyer,H. U.. Gaweln, K., Klotzscb.H
Krebs,H.'A., Williamson,D.H Biochen J
toz, 423-431 (1967)
H .. N 4el l .nb) J D
W i l l :.mson. D
lh)drcrybutymle. ln Mahod ol En.vtu|i.
A ral t" i r ,edi red by B erymeyer' H U .\
AcademicPrcss,New Yo*. (1974)
H .rJno.Y . K o.ugi K . H l osu.T.. U no. 5
Y . C /i a. afti a 4 Id
I(hi kasJ.Y ., S hrgera.
(1983)
t5l , 177-183
Haftno. \ DiabapsCa,p I J. 522-)14 LaoO\
Mrrk" . A R . Mcl nr)F. J.O..D unmn.TM
H. Temns..P. Flei'her'
LrdjumenFBromage
S. J. Bbl Chm26'7,15459-15463(1992)
ISBN:983-2643-15-5
:Pnceerlinss oJ laterndtiah.l Conlekhce On Chemicol a\l Bi.ptuces F.rynl4rin1
2-'
2e' 44utr.A A t U a,pahMrl a),i aS "b-\. K utr L aoau
t6l
i7l
l8l
[9]
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VitanintatulHormones'58'
265