Biochemical and S uc u al Cha ac e iza ion o
C yp ospo idium pa um Lac a e Dehyd ogenase
William J. Cooka*, Olga Senko ichb*, Agus in He nandezc, Haley Speedb, Debasish
Cha opadhyayb,d †
aDepa men o Pa hology, bCen e o Biophysical Sciences and Enginee ing, Uni e si y o
Alabama a Bi mingham, Bi mingham, AL 35294
cIns i u o de Bioquímica Vege al y Fo osin esis (CSIC/U. Se illa), A da. Ame ico Vespucio
49, Se ille 41092, Spain
Depa men o Medicine, Uni e si y o Alabama a Bi mingham, Bi mingham, AL 35294
† Co esponding au ho : debasish@ uab.edu
Phone: (205) 934-0124
Fax: (205) 934-0480
*These au ho s con ibu ed equally
Keywo ds: C ys al s uc u e, lac a e dehyd ogenase, C yp ospo idium pa um
*Manusc ip
Click he e o iew linked Re e ences
Abs ac
The p o ozoan pa asi e C yp ospo idium pa um causes wa e bo ne diseases wo ldwide.
The e is no e ec i e he apy o C. pa um in ec ion. The pa asi e depends mainly on
glycolysis o ene gy p oduc ion. Lac a e dehyd ogenase is a majo egula o o glycolysis.
This pape desc ibes he biochemical cha ac e iza ion o C. pa um lac a e dehyd ogenase
and high esolu ion c ys al s uc u es o he apo-enzyme and ou e na y complexes. The
e na y complexes cap u e he enzyme bound o NAD/NADH o i s 3-ace ylpy idine analog
in he co ac o binding pocke , while he subs a e binding si e is occupied by one o he
ollowing ligands: lac a e, py u a e o oxama e. The esul s e eal dis inc i e ea u es o he
pa asi ic enzyme. Fo example, C. pa um lac a e dehyd ogenase p e e s he ace ylpy idine
analog o NADH as a co ac o . Mo eo e , i is sligh ly less sensi i e o gossypol inhibi ion
compa ed wi h mammalian lac a e dehyd ogenases and no inhibi ed by excess py u a e.
The ac i e si e loop and he an igenic loop in C. pa um lac a e dehyd ogenase a e
conside ably di e en om hose in he human coun e pa . S uc u al ea u es and
enzyma ic p ope ies o C. pa um lac a e dehyd ogenase a e simila o enzymes om ela ed
pa asi es. S uc u al compa ison wi h mala e dehyd ogenase suppo s a common ances y o
he wo genes.
1. In oduc ion
The apicomplexan pa asi e C yp ospo idium pa um causes wa e bo ne diseases and
poses a h ea o wa e supplies wo ldwide (Lei ch & He, 2012; Shi ley e al., 2012). The
pa asi e in ec s a wide spec um o hos s including humans and o he mammals (San in,
2013). In de eloping coun ies, C yp ospo idium is a signi ican cause o dia hea,
con ibu ing o malnu i ion in child en. Al hough in ec ion causes sel -limi ed dia hea in
heal hy adul s, symp oms can be se ious, long las ing, and o en le hal in
immunocomp omised indi iduals (Lei ch & He, 2012; Shi ley e al., 2012). Cu en ly, he e
is no sa is ac o y chemo he apy o accine agains c yp ospo idiosis.
One o he majo impedimen s o iden i ying he apeu ic a ge s in C yp ospo idium is
he lack o knowledge abou i s biochemical and me abolic pa hways. Genome sequencing
indica es ha C. pa um does no ha e a unc ional mi ochond ion and lacks an ac i e K ebs’
cycle (Ab ahamsen e al., 2004) bu encodes all glycoly ic enzymes. Since he pa asi e
appea s o depend p ima ily on anae obic oxida ion o glucose o ene gy me abolism
(Coombs, 1999, Den on e al., 1996), enzymes in he glycoly ic pa hway and hose ha play
egula o y oles may o e po en ial a ge s o an i-c yp ospo iodial d ugs. S udies wi h
o he apicomplexan pa asi es ha e shown ha inhibi ion o glycolysis may be a use ul
s a egy o an ipa asi ic he apy (Basco e al., 1995; B essi e al., 2000; Dando e al., 2001;
Deck e al., 1998; Wang, 1984). To de elop a de ailed unde s anding o he key egula o y
mechanisms ha a e cha ac e is ic o he pa asi e, we a e s udying a numbe o enzymes ha
con ol glycolysis in C. pa um (Senko ich e al, 2005; Cook e al., 2009; Cook e al., 2012).
Lac a e dehyd ogenase (LDH) plays a cen al ole in egula ing glycolysis. LDH is a key
enzyme o he anae obic espi a ion s ep in which py u a e is educed o lac a e wi h he
concomi an oxida ion o NADH o NAD+ (E e se & Kaplan, 1973). This eac ion is c ucial
o p og ession o glycolysis. Fi s ly, emo al o py u a e allows glycolysis o p og ess in
he di ec ion owa d gene a ing mo e ATP molecules. Secondly, i egene a es NAD+, which
is equi ed o he oxida ion o glyce aldehyde 3-phospha e in glycolysis, a s ep ca alyzed by
he glycoly ic enzyme glyce aldehyde 3-phospha e dehyd ogenase.
In humans he e a e wo majo o ms o LDH. The M- o m is ound p edominan ly in
anae obic issues such as skele al muscle, and he H- o m is p esen mos ly in ae obic issues
such as ca diac muscle. The M- o m and H- o m enzymes a e 78% iden ical in hei p ima y
sequences. Al hough he p ima y sequence o LDH is highly conse ed among a ious
o ganisms, LDHs o a numbe o p o ozoan pa asi es including C. pa um (CpLDH) di e
om human LDH in se e al key amino acid posi ions, and some possess a unique inse ion in
he ac i e si e loop (Fig. 1) (Deck e al., 1998; Gomez e al., 1997; Dunn e al., 1996;
Sessions e al., 1997; Win e e al., 2003). LDHs o ce ain p o ozoa also demons a e
dis inc i e s uc u al and biochemical p ope ies. Fo example, he P. alcipa um enzyme
(P LDH) exhibi s 200-300 old highe ac i i y wi h he NADH analog 3-ace yl py idine
adenine dinucleo ide (APADH) han he human M and H isozymes (Gomez e al., 1997).
Biochemical di e ences be ween human LDH and P LDH ha e been exploi ed o he
de elopmen o a diagnos ic ool o de ec ion o mala ia in he blood o in ec ed indi iduals
(Klene man & Dickson, 1992; Makle & Hin ichs, 1993) and de elopmen o selec i e
inhibi o s o he pa asi ic enzyme (Gomez e al., 1997). Di e ences in he co ac o binding
si es o LDH om human and P. alcipa um allow de i a i es o he na u al p oduc gossypol
o selec i ely inhibi pa asi ic LDHs (Dando e al., 2001; Deck e al., 1998; Roye e al.,
1998). S uc u al analysis o LDH om wo di e en species o plasmodium showed ha
hese enzymes possessed dis inc i e ea u es, which may be use ul o de elopmen o
species-speci ic inhibi o s o he plasmodium enzymes (Dunn e al., 1996; Sessions e al.,
1997; Win e e al., 2003).
He e we desc ibe he esul s o enzyma ic and s uc u al analyses o CpLDH. We
discuss he c ys al s uc u es o he apo-enzyme and ou e na y complexes wi h subs a e,
inhibi o (oxama e) o p oduc bound a he ac i e si e. To isualize he di e ences in he
binding o NAD and APAD a he co ac o binding pocke , s uc u es o py u a e complexes
we e de e mined sepa a ely wi h each co ac o . We also compa e he p ope ies o CpLDH
wi h LDHs om human and apicomplexan pa asi es. Finally, we p esen a compa ison o he
s uc u es o CpLDH and C. pa um mala e dehyd ogenase (CpMDH).
2. Ma e ials and Me hods
2.1. P epa a ion o enzyme
Exp ession and pu i ica ion o CpLDH ha e been desc ibed in de ail (Senko ich e al.,
2005). B ie ly, he ecombinan p o ein was exp essed in E. coli s ain Rose a (DE3)pLysS.
The bac e ial cell pelle was lysed in bu e I (50 mM T is-HCl, 1 mM benzamidine
hyd ochlo ide, 0.1 mM PMSF, 5 mM DTT and 1 mM EDTA, pH 7.4). The esul ing
suspension was subjec ed o cen i uga ion a 20,000 pm o 30 min, and he supe na an was
ea ed wi h 0.2% p o amine sul a e o 20 min a 4C. CpLDH was p ecipi a ed om
cla i ied cell- ee ex ac by adding ammonium sul a e o 40% sa u a ion. The esuspended
pelle was dialyzed o e nigh in bu e I and applied o a Supe dex 200 size exclusion column
(Ame sham Pha macia). As epo ed p e iously, enzyma ically ac i e CpLDH elu ed om
he Supe dex 200 column as a e ame as calcula ed om he elu ion olume and = nea ly
homogeneous pu i ied p o ein mig a ed as a majo band o app oxima ely 33 kDa on SDS-
PAGE (Senko ich e al., 2005). F ac ions con aining enzyma ically ac i e CpLDH we e
pooled, dialyzed agains bu e II (50 mM T is-HCl, 1 mM EDTA and 5 mM DTT, pH 8.0)
and applied o a Mono Q 10/10 column (Ame sham Pha macia). A e washing he column
wi h bu e II, he bound p o ein was elu ed using a linea g adien o sodium chlo ide (0-0.3
M) in 15 column olumes o bu e II. Pu i ied p o ein was concen a ed by ul a il a ion o a
inal concen a ion o 21 mg/ml. Sequencing o he ecombinan plasmid e ealed ha he
DNA encodes alanine a posi ion 202, lysine a posi ion 265 and glu ama e a posi ion 311
(see Foo no e o explana ion o LDH numbe ing), while he da abase sequence o CpLDH
(GenBank accession No. AF274310) has aline, a ginine and lysine, espec i ely, a hese
posi ions (Fig. 1). Simila esul s we e ob ained in mul iple PCR expe imen s. We do no
know i he sequence di e ences a e PCR e o s, mu a ions co esponding o DNA
polymo phism, o i he e a e e o s in he GenBank da abase.
2.2. Enzyme ac i i y
LDH enzyme assays we e pe o med in a empe a u e-con olled UV-Visible
spec opho ome e (DU740, Beckman Ins umen s) using s anda d me hods (Dando e al.,
2001). Reac ion eloci ies we e measu ed a 25C o 1 min by ollowing dec ease o
inc ease in abso bance a 340 nm due o oxida ion o NADH o educ ion o NAD+. The
CpLDH ac i i y in he di ec ion o educ ion o py u a e o lac a e was measu ed in 50 mM
sodium ace a e bu e , pH 5.5, and ac i i y in he di ec ion o oxida ion o lac a e o py u a e
was measu ed in 50 mM T is-HCl bu e , pH 9.1. A ypical eac ion mix u e con ained
subs a e (10 M - 5 mM py u a e o 50 μM - 20 mM lac a e) and co ac o (3 - 150 μM
NADH o 10 - 600 μM NAD+); enzyme eac ion was ini ia ed by adding 1 - 5 μg o pu i ied
CpLDH. The pH dependence o he CpLDH ac i i y was de e mined using h ee bu e s wi h
o e lapping pHs: 50 mM sodium ace a e, pH 3.6 - 6.0, 50 mM sodium phospha e, pH 5.6 -
7.2 and 50 mM T is-HCl, pH 6.6 - 9.5. When APAD+/APADH was used as co ac o ,
eac ion eloci ies we e measu ed by ollowing inc ease o dec ease in abso bance a 363 nm
due o educ ion o APAD+ o oxida ion o APADH. The kine ic pa ame e s (Michaelis
cons an Km, maximum eloci y Vmax and u no e a e Kca ) o subs a es and co ac o s and
he inhibi o y cons an Ki alue o gossypol we e de e mined by non-linea eg ession using
ANEMONA Excel empla es (He nandez and Ruiz, 1998).
2.3. C ys alliza ion and da a collec ion
CpLDH was c ys allized unde se e al di e en condi ions a 4C. As epo ed
p e iously c ys als o he apo enzyme sui able o da a collec ion we e g own om 16.5%
( / ) PEG 2000, 0.1 M T is-HCl (pH 7.0) and 0.08% n-oc yl-β-D-glucopy anoside
(Senko ich e al., 2005). Fo c ys alliza ion o he e na y complexes, he concen a ed
p o ein was incuba ed wi h 1 mM subs a e (py u a e, lac a e o oxama e) and 100 μM
NADH, NAD+ o APAD+ o an hou on ice. C ys als o he e na y complexes we e
ob ained a 4C using 1.45-1.65 M ammonium sul a e in 0.1 M sodium cacodyla e (pH 6.75
o 7.25).

X- ay di ac ion da a we e collec ed unde c yogenic condi ions (-180C) using a
c yop ese a i e solu ion con aining 25% glyce ol in he ese oi solu ion. In ensi y da a
we e p ocessed using Denzo and Scalepack (O winowski & Mino , 1997). C ys als o apo
CpLDH belong o space g oup P3212 and con ain ou monome s ( wo dime s) in he
asymme ic uni . C ys als o he e na y complexes belong o space g oup P3221 and con ain
wo monome s (one dime ) in he asymme ic uni .
2.4. S uc u e de e mina ion and e inemen
The c ys al s uc u e o he e na y complex o CpLDH wi h py u a e and APAD+ was
sol ed by molecula eplacemen using he CNS p og am package (B ünge e al., 1998) wi h
da a in he ange 20 - 4.5 Å o he o a ion and ansla ion sea ches. Calcula ion o a sel -
o a ion unc ion e ealed a s ong peak a kappa = 180º, indica ing he p esence o a
nonc ys allog aphic wo- old axis. Using a sea ch model o CpLDH buil om P LDH
(PDBID 1T2D), posi ions o he wo monome s in he asymme ic uni we e de e mined.
The co ec enan iomo phic space g oup (P3221) was de e mined om he alues o
co ela ion coe icien and R ac o in he molecula eplacemen ials wi h he wo
enan iomo phic space g oups. Re inemen o he s uc u e was pe o med ini ially by
simula ed annealing using CNS wi h he s e eochemical pa ame e iles de ined by Engh and
Hube (1991). No sigma cu o was applied o he da a. Ten pe cen o he da a was
andomly selec ed and emo ed p io o e inemen o analysis o he ee R ac o . The wo
subuni s in he asymme ic uni we e es ained by he non-c ys allog aphic symme y
h oughou he simula ed annealing e inemen . The molecula model was imp o ed by
cycles o manual i ing o 2Fo-Fc elec on densi y maps using he p og am COOT (Emsley
e al., 2010) al e na ing wi h e inemen .
An Fo-Fc elec on densi y map allowed placemen o APAD+ and py u a e in each
monome . As he e inemen p og essed, wa e molecules we e added by using he wa e -
picking ou ines in CNS and COOT. All wa e molecules we e e i ied by inspec ion o he
maps. In he la e s ages o e inemen , nonc ys allog aphic symme y es ain s we e
emo ed, and he es ained e inemen op ion in REFMAC5 (Mu shudo e al., 2011) was
used.
The s uc u es o he apo enzyme and he o he h ee e na y complexes we e sol ed by
molecula eplacemen using he LDH dime om he C. pa um LDH/APAD+/py u a e
s uc u e as he sea ch model. The e inemen p ocedu e was he same in each case as
desc ibed abo e. A omic coo dina es and s uc u e ac o s ha e been deposi ed in he P o ein
Da a Bank wi h PDBIDs 4ND1 (NAD+/oxama e complex), 4ND2 (APAD+/py u a e
complex), 4ND3 (NADH/lac a e complex), 4ND4 (NAD+/py u a e) and 4ND5 (apo
enzyme)†.
3. Resul s
As epo ed p e iously pu i ied ecombinan CpLDH elu ed as a e ame as e ealed by size
exclusion ch oma og aphy (Senko ich e al., 2005). On SDS PAGE analysis he pu i ied
p o ein mig a ed as a single band o app oxima ely 33 kDa.
3.1. Enzyme ac i i y
Using bu e solu ions o o e lapping pH, we de e mined pH op ima o LDH eac ions
in bo h di ec ions. The op imal pH o he educ ion o py u a e was 5.0 - 5.5, bu he
op imal pH o he oxida ion o lac a e was 9.0 - 9.5 (Fig. 2A and B). In Table 1, kine ic
pa ame e s o CpLDH a e compa ed wi h hose o human iso o ms, P. alcipa um and T.
gondii LDH. Like P LDH, CpLDH p e e s APAD+ and APDH o e NAD+ and NADH,
espec i ely, as a co ac o . The kca o CpLDH was wo- old g ea e o APADH han o
NADH. Simila ly, he enzyma ic ac i i y o CpLDH was ~5 imes highe wi h APAD+ han
wi h NAD+ as a co ac o . On he o he hand, he human enzymes show ~17-20 old highe
ac i i y wi h NAD+ as compa ed wi h APAD+. The e o e, wi h APAD+ he ac i i y o
CpLDH is 100 old highe han he human coun e pa s. In compa ison P LDH is 200 imes
mo e ac i e wi h APAD+ han he human enzyme (Gomez e al., 1997). In he case o
CpLDH a small bu signi ican nega i e coope a i i y owa ds py u a e was obse ed (Hill
coe icien h= 0.69±0.00). Howe e , in he case o NADH, he es ima ed coope a i i y (Table
1) was no signi ican since he sum o leas -squa es a e i ing he da a o a Hill model was
nea ly iden ical o ha obse ed a e a pu e Michaelis-Men en i (da a no shown). On he
o he hand, a nega i e coope a i i y was obse ed when APAD+ was used as a co ac o .
Gossypol and a numbe o i s de i a i es a e known o inhibi LDH ac i i y (Deck e al.,
1998; Conne s e al., 2005). As seen wi h o he LDHs (Olgia i & Toscano, 1983), he
† PDBIDs a e shown in i alics h oughou his manusc ip
mechanism o inhibi ion o CpLDH by gossypol is compe i i e wi h NADH (Fig. 2C). The
obse ed Ki alue o gossypol (11.6 µM) o CpLDH is in he same ange bu sligh ly highe
han o he human enzyme (1.9 and 1.4 µM o M and H- o ms, espec i ely; Gomez e al.,
1997).
In gene al, LDHs a e inhibi ed by excess py u a e, p esumably due o he o ma ion o
an NAD+/py u a e complex (Wang, 1977). A dis inc i e biochemical ea u e o some
p o ozoan LDHs is hei insensi i i y o high py u a e concen a ion. As shown in Fig. 2D,
CpLDH does no display any measu able inhibi ion o py u a e concen a ions up o a leas
20 mM.
3.2. Gene al desc ip ion and quali y o he s uc u es
C ys al s uc u es o CpLDH we e de e mined in he apo o m and as e na y complexes
con aining he ollowing: py u a e and NAD+, lac a e and NADH, oxama e and NAD+,
py u a e and APAD+. S a is ics o da a collec ion a e shown in Table 2. Re inemen
s a is ics and Molp obi y analyses (Da is e al., 2007) a e lis ed in Table 3.
3.2.1. Apo CpLDH
Apo CpLDH c ys allizes in he space g oup P3212, and he c ys al s uc u e con ains wo
dime s ( wo hal e ame s) in he asymme ic uni . The c ys allog aphic symme y pai s o
each dime o m he unc ional e ame s. The model includes esidues 17-98 and 112-330
o monome A, 17-98 and 112-329 o monome B, 18-98 and 112-329 o monome C, and
18-98 and 112-327 o monome D. The elec on densi y o C- e minal esidues and he
ac i e si e loop ( esidues 99-111) was ex emely weak, and hose esidues could no be
modeled. The ou CpLDH monome s in he asymme ic uni a e nea ly supe imposable.
Compa ed o monome A, he oo mean squa e de ia ion o all Cα-a oms is 0.327 Å o B
o A, 0.353 Å o C o A and 0.332 Å o D o A.
3.2.2. Te na y complexes o CpLDH
C ys als o he ou e na y complexes belong o space g oup P3221.The asymme ic uni
con ains wo monome s ela ed by non-c ys allog aphic 2- old symme y (Fig. 3A). The
comple e e ame is o med wi h hei symme y pa ne s ela ed by a c ys allog aphic 2- old
axis. In each complex he model includes esidues 17-333 o each chain (see no e a he end
o he manusc ip ); only he las ou C- e minal esidues we e no isible in he elec on-
densi y maps. The a angemen o he monome s in he e ame is simila o ha seen in
o he LDH s uc u es. The wo monome s in he asymme ic uni o m he majo in e ace
and bu y app oxima ely 7900 Å2 o su ace a ea. Supe posi ion o all ou complex
s uc u es, using monome A o he CpLDH/NAD+/py u a e s uc u e as he e e ence,
esul s in .m.s.d. alues o 0.113 Å o CpLDH/NADH/lac a e, 0.176 Å o CpLDH/NAD+/
oxama e, and 0.107 Å o CpLDH/APAD+/py u a e.
As shown in Supplemen a y Fig. S1, in all ou e na y complexes he NAD+/NADH si e
is ully occupied in bo h monome s in he asymme ic uni . Elec on densi y o subs a e
molecules was also excellen in each subuni o all complexes excep in he NADH/lac a e
complex, in which he densi y o lac a e was clea only in subuni A. A e age B- ac o s o
he co- ac o and subs a e/analog molecules a e compa able o hose o he p o ein esidues
in each complex (Table 3). In all cases he CpLDH monome s in he asymme ic uni and he
bound co ac o s a e nea ly supe imposable. The oo mean squa e de ia ion be ween
monome s A and B o all 317 Cα a oms is 0.278 Å o CpLDH/NAD+/py u a e, 0.465 Å o
CpLDH/NADH/lac a e, 0.379 Å o CpLDH/NAD+/oxama e, and 0.347 Å o
CpLDH/APAD+/py u a e.
The o e all quali y o he s uc u es o CpLDH is excellen . Only Ala164 and Gly283 in
each monome exhibi phi, psi angles in non-allowed egions o he Ramachand an plo . The
esidue co esponding o Ala164 is glycine in human and plasmodium LDH. All o he
s uc u es con ain se e al glyce ol molecules associa ed wi h each chain.
3.3. NADH/APADH and subs a e binding si es
In he ollowing desc ip ion monome A om he e na y complex
CpLDH/NAD+/py u a e (4ND4) will be used as he empla e unless o he wise s a ed.
CpLDH monome s a e composed o wo domains wi h he ac i e si e loca ed a he in e ace
o he wo domains (Fig. 3A). The NAD-binding domain (shown in ligh pink in Fig. 3A) is
cha ac e ized by a ypical Rossmann old consis ing o a six-s anded pa allel β-shee lanked
by α-helices. As shown in Fig. 3B and C, he adenine ing o he co ac o lies in a
hyd ophobic pocke o med by Ile27, Phe52, Ile54, Ty 85, Ala98, Ile119 and Val123 and
packs agains he side chains o Ile54 and Ala98. The hyd oxyl oxygen a oms O2B and O3B
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Figu e legends 1
Figu e 1. Sequence alignmen 2
P ima y sequences o LDH om a ious o ganisms we e aligned based on s uc u al 3
homology. The labelling o seconda y s uc u al elemen s co esponds o he CpLDH 4
s uc u e. The h ee black iangles indica e changes in he CpLDH sequence compa ed o 5
he GenBank sequence. This igu e was p epa ed using ESP ip (Goue e al., 1999). 6
Figu e 2. Enzyma ic ac i i y 7
A. CpLDH ac i i y o educ ion o py u a e was measu ed using bu e solu ions a 8
di e en pH. Rela i e ac i i y is plo ed agains pH alues. 9
B. CpLDH ac i i y o oxida ion o lac a e was measu ed using bu e solu ions a 10
di e en pH. Rela i e ac i i y is plo ed agains pH alues. 11
C. Ki alues o gossypol we e de e mined o educ ion o py u a e wi h NADH a pH 12
5.5. Py u a e concen a ion was 5 mM, and NADH concen a ions we e a ied 13
be ween 5-35 µM. Gossypol concen a ions we e 0, 2.5, 7.5 and 10 µM. 14
D. CpLDH ac i i y was de e mined a sa u a ing concen a ion o NADH (150 M) and 15
a ying concen a ions o py u a e by measu ing he dec ease in abso bance a 340 nm. 16
Figu e 3. Assembly o CpLDH and de ails o he co ac o binding pocke 17
A. Ca oon d awing showing assembly o CpLDH in he asymme ic uni o he e na y 18
complex wi h NAD+ and py u a e. The wo monome s comp ising he asymme ic uni 19
a e ela ed by a nonc ys allog aphic 2- old axis ha is app oxima ely pe pendicula o 20
he page. Ca aly ic and NAD-binding domains o monome A a e depic ed in magen a 21
and ligh pink, espec i ely. The helix connec ing he wo domains is shown in ed and 22
ma ine blue in he wo subuni s. Ca aly ic and NAD-binding domains o he B subuni 23
a e colo ed cyan and ligh cyan, espec i ely, and he connec ing helix is shown in 24
sla e. NAD+ and py u a e a e shown as s ick models. 25
B. Close-up iew o he NAD binding si e in CpLDH, colo ed as in Figu e 3(A). NAD is 26
shown as a s ick model (ca bon: whi e). CpLDH esidues in he NAD-binding si e a e 27
also shown as s ick models (ca bon: ose o magen a). 28

C. Su ace d awing showing a close-up iew o he adenine binding pocke in CpLDH. 1
NAD is shown as a s ick model (ca bon: g een). CpLDH esidues lining he pocke a e 2
shown as s ick models (ca bon: whi e) in he semi- anspa en su ace diag am. 3
D. S e eoscopic iew o he NAD-binding si e in he CpLDH/NAD+/py u a e complex. 4
NAD (ca bon: g een), py u a e (ca bon: yellow) and he CpLDH esidues (ca bon: 5
ose) o ming hyd ogen bonds o NAD and py u a e a e shown. A g171 and His195 6
a e also shown in s ick model (ca bon: magen a). Po en ial hyd ogen bonds a e 7
illus a ed in do ed lines. Two wa e molecules nea NAD a e shown as ed sphe es. 8
Figu e 4. Changes in CpLDH s uc u e upon subs a e and co ac o binding 9
A. Ca oon d awing showing supe posi ion o he s uc u es o apo CpLDH (magen a) and 10
he e na y complex (cyan) wi h NAD+/py u a e, highligh ing he a eas ha show 11
majo changes. These a eas a e shown in blue on he complex s uc u e, and he amino 12
acid esidues a e labeled. The ac i e si e loop obse ed in he complex ( esidues 99-13
111, colo ed deep blue) is diso de ed in he apo- o m. NAD (ca bon: g een) and 14
py u a e (ca bon: yellow) a e shown as s ick models. 15
B. Close-up iew o he egion encompassing esidues 138-145 o CpLDH in he apo and 16
e na y complex s uc u es. Amino acid esidues a e shown as s ick models: apo 17
(ca bon: ose) and complex (ca bon: cyan). 18
C. Close-up iew o he egion encompassing esidues 194-198 o CpLDH in he apo and 19
e na y complex s uc u es. Amino acid esidues a e shown as s ick models: apo 20
(ca bon: ose) and complex (ca bon: cyan). The His195 side chain is o ien ed owa ds 21
he subs a e in he complex. 22
D. Close-up iew o he egion encompassing esidues 234-245 o CpLDH in he apo and 23
e na y complex s uc u es. Amino acid esidues a e shown as s ick models: apo 24
(ca bon: ose) and complex (ca bon: cyan). In he complex T p236 mo es close o he 25
subs a e. 26
Figu e 5. Compa ison o CpLDH wi h human LDH. 27
A. Supe posi ion o A monome s o he e na y complex CpLDH/NAD+/oxama e (cyan) 28
and human LDH wi h NADH and oxama e (magen a) (1I0Z; Read e al., 2001). NAD+ 29
in CpLDH is shown as a s ick model (ca bon: g een), and oxama e is shown as a ball 30
and s ick model (ca bon: yellow). Th ee loop egions ha show signi ican di e ences 1
in con o ma ion a e labeled on he CpLDH ca oon. 2
B. De ailed iew o he loops co e ing he en ance o he ac i e si e. The labelled esidues 3
a e om CpLDH. (2Fo-Fc elec on densi y con ou ed a 1.2 o he esidues in he wo 4
loops nea he ac i e si e is displayed in supplemen a y igu e). 5
C. De ailed iew o he an igenic loops in CpLDH (s ick model) and human LDH ( ed). 6
(2Fo-Fc elec on densi y con ou ed a 1.2 o he loop esidues in CpLDH is shown in 7
supplemen a y igu e). 8
Figu e 6. Compa ison o CpLDH s uc u e wi h P LDH and TgLDH 9
A. Ca oon diag am showing supe posi ion o CpLDH s uc u e (cyan) wi h P LDH 10
(yellow) and TgLDH (whi e) s uc u es. S uc u es o he CpLDH NAD+/py u a e 11
complex (4ND1), TgLDH/APAD/oxala e complex (1PZF) and P LDH/NAD/oxala e 12
complex (1T2D) we e used o supe posi ion. NAD and py u a e molecules in CpLDH 13
a e shown as s ick models. 14
B. A close up iew showing he di e ences in he con o ma ion nea he ac i e si e o 15
CpLDH. Residues 101-102 ha a e di e en in CpLDH as compa ed o o he 16
apicomplexan LDHs a e shown as s ick models. The con o ma ion o he loop 17
comp ising esidues 243-246 is also di e en in CpLDH. 18
Figu e 7. Compa ison o CpLDH, P LDH and TgLDH wi h CpMDH 19
A. Supe posi ion o one monome o CpLDH (4ND1, cyan) and CpMDH (2HJR, yellow) 20
Only he ac i e si e egion is shown because he di e ences a e es ic ed o his a ea. 21
Subs a e binding esidues o CpLDH and co esponding esidues o CpMDH a e 22
shown as s ick models (ca bon a oms a e colo ed same as he espec i e p o ein chain). 23
CpLDH numbe ing is used o labeling excep esidue A g94 o CpMDH, which is 24
shown hyd ogen bonded o ci a e in he ac i e si e o CpMDH. 25
B. Di e ences in he ac i e si e egions o CpLDH (blue), P LDH (magen a) and TgLDH 26
(g ey) compa ed o CpMDH (yellow). Glu amine 102 se es as he speci ici y esidue 27
o canonical LDHs. In all MDHs he co esponding esidue is an a ginine (A g94 in 28
CpMDH). In apicomplexan LDHs, esidue 102 is lysine (Lys102 in TgLDH). In 29
P LDH and TgLDH he e is a i e esidue inse ion in he ac i e si e loop. Due o he 30
inse ion, esidue T p107 becomes he speci ici y esidue in P LDH and CpLDH. 31
Howe e , LDHs o C yp ospo idium a e excep ional among apicomplexan LDHs in 1
ha esidue 102 in CpLDH is glycine. Residue 103 is an a ginine in CpLDH, and i 2
emains excluded om he ac i e si e. 3
Figu e 8. Hyd ogen bonding in e ac ions in he ac i e si e o LDH 4
A. Human LDH NAD+/oxama e complex (1I0Z, Read e al., 2001) S ick diag am showing 5
amino acid esidues and wa e molecules o ming hyd ogen bonds wi h NAD and 6
oxama e. 7
B. CpLDH NAD+/oxama e complex (4ND1). Amino acid esidues in ol ed in hyd ogen 8
bonding in e ac ions wi h NAD and oxama e. 9
Figu e 9. Compa ison o NAD+ and APAD+ binding in CpLDH. The APAD+ ca bon a oms 10
a e ligh g een; he NAD+ ca bons a e ligh magen a. Impo an esidues in he ac i e si e 11
a e included as s ick models. 12
13
14
Table 1. Compa ison o kine ic pa ame e s o CpLDH, TgLDHs, P LDH and human LDHs
CpLDH
P LDH
Hs_M
Hs_H
TgLDH1
TgLDH2
Km
Kca
Kca /Km
h
Km
Kca
Km
Kca
Km
Kca
Km
Kca
Km
Kca
Py u e
197.3±0.1
124.0±5.6
0.63±0.04
0.69±0.00
30
27
170
18
55
13
120
35
800
4.6
Lac a *
1.8±0.2
2.7±0.3
0.002±0.141
N.D.
12.0
17
11.0
4.9
13.0
3
8.0
1.6
54.0
5.8
NADH
16.9±0.0
125.2±3.9
7.41±0.03
1.23±0.03
7
27
5
18
8
13
4.2
37
1.9
4.3
APAH
16.6±0.0
231.7±7.2
13.96±0.03
1.08±0.06
NAD+
30.3±0.0
3.3±0.1
0.11±0.03
1.04±0.07
86
17
93
4.9
37
3
348
1.3
640
0.45
APAD+
8.5±0.1
14.9±0.8
1.75±0.05
0.69±0.08
123
51
56
0.24
37
0.17
50
34
78
13
Uni s: Km in M, excep o lac a e* (mM); Kca in min-1 ×10-3; Kca /Km in M-1 min-1 ×10-3.
Km: Concen a ion o subs a e a which he ca aly ic a e is hal -maximal; Kca : numbe o subs a e molecules p ocessed pe molecule
o enzyme pe uni o ime. Kca /Km: speci i y cons an ; h :Hill coe icien (coope a i i y); N.D.: no de e mined.
Da a o TgLDH aken om Dano e al., 2001; o P LDH and human LDHs (Hs_H and Hs_M) a e aken om Gomez e al., 1997.
Hs_H and Hs_M e e o he H and M iso o ms.
Table 1
C

Ac i i y, mic omole/min/mg
D
50
40
30
20
10
0
0 5 10 15 20 25 30
[Py u a e], mM
Figu e 3
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Figu e 4
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Figu e(s)
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Figu e 6
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Figu e 7
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Figu e 8
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Figu e 9
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Enzyma ic cha ac e iza ion and c ys allog aphic analysis C yp ospo idium pa um Lac a e
dehyd ogenase e eal dis inc i e ea u es o he pa asi ic enzyme and sugges ha i is an
excep ional membe in he apicomplexan lac a e dehyd ogenase amily.
Highligh s ( o e iew)