Bioscience Repo s (2020) 40 BSR20191806
h ps://doi.o g/10.1042/BSR20191806
Recei ed: 29 May 2019
Re ised: 17 Decembe 2019
Accep ed: 19 Decembe 2019
Accep ed Manusc ip online:
20 Decembe 2019
Ve sion o Reco d published:
10 Janua y 2020
Resea ch A icle
Insigh s in o he Cnx1E ca alyzed MPT-AMP
hyd olysis
Thomas W. He che 1, Joe n K ausze1, S en Ho meis e 1, Dagma Zwe schke1, Thomas Lindel2,
Wul Blanken eld 3,4, Ral R. Mendel1and Tobias K use1
1TU B aunschweig, Ins i u e o Plan Biology, Spielmanns asse 7, 38106 B aunschweig, Ge many; 2TU B aunschweig, Ins i u e o O ganic Chemis y, Hagen ing 30, 38106
B aunschweig, Ge many; 3S uc u e and Func ion o P o eins, Helmhol z Cen e o In ec ion Resea ch, Inho ens asse 7, 38124 B aunschweig, Ge many; 4TU B aunschweig,
Depa men o Bio echnology, Ins i u e o Biochemis y, Bio echnology and Bioin o ma ics, Spielmanns asse 7, 38106 B aunschweig, Ge many
Co espondence: T. K use ([email p o ec ed])
Molybdenum inse ases (Mo-inse ases) ca alyze he inal s ep o molybdenum co ac o
(Moco) biosyn hesis, an e olu iona y old and highly conse ed mul i-s ep pa hway. In he
i s s ep o he pa hway, GTP se es as subs a e o he o ma ion o cyclic py anop e in
monophospha e, which is subsequen ly con e ed in o molybdop e in (MPT) in he second
pa hway s ep. In he ollowing syn hesis s eps, MPT is adenyla ed yielding MPT-AMP ha
is subsequen ly used as subs a e o enzyme ca alyzed molybda e inse ion. Molybda e in-
se ion and MPT-AMP hyd olysis a e ca alyzed by he Mo-inse ase E-domain. Ea lie wo k
epo ed a highly conse ed aspa a e esidue o be essen ial o Mo-inse ase unc ional-
i y. In his wo k, we con i med he mechanis ic ele ance o his esidue o he A abidopsis
haliana Mo-inse ase Cnx1E. We ound ha he conse a i e subs i u ion o Cnx1E esidue
Asp274 by Glu (D274E) leads o an a es o MPT-AMP hyd olysis and hence o he accumu-
la ion o MPT-AMP. We u he showed ha he MPT-AMP accumula ion goes in hand wi h
he accumula ion o molybda e. By c ys alliza ion and s uc u e de e mina ion o he Cnx1E
a ian D274E, we iden i ied he po en ial eason o he missing hyd olysis ac i i y in he
diso de o he egion spanning amino acids 269 o 274. We easoned ha his is caused by
he inabili y o a glu ama e in posi ion 274 o coo dina e he oc ahed al Mg2+-wa e complex
in he Cnx1E ac i e si e.
In oduc ion
The molybdenum co ac o (Moco) biosyn hesis pa hway in ol es he conce ed ac ion o nume ous en-
zymes ha a e conse ed h oughou all kingdoms o li e [1,2]. The ini ial subs a e o Moco biosyn-
hesis is GTP, which is con e ed in o cyclic py anop e in monophospha e (cPMP) in he i s eac ion
o a mul i-s ep pa hway [2]. This eac ion in ol es adical S-adenosyl me hionine chemis y and is ca -
alyzed by he cPMP syn hase [3]. In he ollowing s ep o Moco biosyn hesis, cPMP is con e ed in o
molybdop e in (MPT), a eac ion ha is dis inguished by he in oduc ion o he di hiolene mo i cha ac-
e is ic o MPT. This eac ion is ca alyzed by he he e o e ame ic MPT-syn hase complex, which com-
p ises wo la ge and wo small subuni s [4,5]. He e, he wo la ge subuni s o m he co e o he complex,
while each o he wo small subuni s in e ac s independen ly wi h one o he la ge subuni s [5]. Molyb-
da e is inse ed in o he MPT backbone in a subsequen s ep, yielding Moco. In i o in e ac ion s udies
e ealed he plan MPT syn hase complex and he molybdenum inse ase (Mo-inse ase) o in e ac wi h
each o he , hus p o iding he amewo k necessa y o e icien , p o ec ed and di ec ed me aboli e ans-
e [6]. The molybda e inse ion eac ion in ol es bo h unc ional domains o Mo-inse ases, namely E-
and G-domain [7] whose ole o he Mo-inse ion eac ion has been s udied in de ail using he plan
(A abidopsis haliana)Mo-inse aseCnx1asmodelenzyme[8–15].No ably, hesedomainsa e eac-
i e as sepa a ely exp essed domains (p oka yo es) o used oge he (euka yo es, excep he lowe alga
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Chlamydomonas einha d ii [16]). Ini ially, he Mo-inse ase G-domain ca alyzes he liga ion o an AMP molecule
o he e minal phospha e g oup o MPT, yielding adenyla ed MPT (MPT-AMP, [11,12]). Subsequen ly, MPT-AMP is
ans e ed o he Mo-inse ase E-domain, which equi es molybda e o be bound o he E-domain oxo-anion en y
si e [15]. Wi hin he Cnx1E ac i e si e, MPT-AMP adop s a con o ma ion ha is di e en om he one ound in he
Cnx1G MPT-AMP co-s uc u e [11] and ha p ope ly o ien s he di hiolene mo i owa d enzyme bound molybda e
[14,15]. Subsequen ly he MPT-AMP phospho ic anhyd ide bond is hyd olyzed, a eac ion ha is belie ed o be he
p e equisi e o enzyme ca alyzed molybda e inse ion in o he MPT di hiolene mo i [13–15]. Recen wo k sugges ed
ha he Cnx1E ca alyzed molybda e inse ion eac ion in ol es he eloca ion o molybda e om he ini ial oxo-anion
binding si e o he inse ion si e [15]. Upon syn hesis Moco is ans e ed o he cellula use enzymes and/o o he
cellula Moco ans e /s o age sys em [2,6,17,18]. Wi hin his wo k we desc ibe he iden i ica ion and, o he i s
ime, he biochemical cha ac e iza ion o a hyd olysis inac i e Cnx1E a ian (Cnx1E D274E) ha may pa e he way
o deciphe he molecula mechanism(s) unde lying Cnx1E eac i i y.
Ma e ials and me hods
Gene a ion o Cnx1E a ian D274E
Cnx1E a ian D274E was gene a ed ollowing he QuikChange (Agilen Technologies) p o ocol modi ied o he
use o Phusion®High-Fideli y DNA Polyme ase (The mo Fische ). As desc ibed ea lie [15], we used he Cnx1E
wild- ype pGPlus exp ession ec o [14] as empla e o PCR-based mu agenesis. The sequence o he p ime
pai used o in oduce he single amino acid exchange D274E was 5´-gggagacagggaG cg caagcca ac cgaag-3´ and
5´-ag aa ggc gacgaaC ccc g c ccca ga-3´ (misma ches in uppe case). The iden i y o he gene a ed cons uc was
subsequen ly con i med by sequencing.
Exp ession and pu ifica ion o ecombinan Cnx1E
Fo c ys alliza ion expe imen s, ecombinan Cnx1E was exp essed and pu i ied as desc ibed p e iously [15]. Fo
ecombinan biochemis y, Cnx1E was exp essed and pu i ied as desc ibed p e iously [14].
In i o ans e o MPT-AMP on Cnx1E
In i o ans e o MPT-AMP on Cnx1E was essen ially ca ied ou as desc ibed p e iously [14].
HPLC-based quan ifica ion o Cnx1E bound MPT-AMP and Moco/MPT
The p o ocol desc ibed he e was adap ed and modi ied om p e ious p o ocols [11,15,19,20] and used syn he ic de-
phospho Fo mA [21] o Moco/MPT quan i ica ion in he biological samples. To quan i y Cnx1E bound MPT-AMP
and Moco/MPT, he p o ein p epa a ions we e p ocessed di ec ly a e elu ion om he S ep-Tac in®Supe low®
high-capaci y esin (IBA) and p io o concen a ing he sample hus ensu ing minimal deg ada ion o p o ein bound
Moco/MPT and/o MPT-AMP, espec i ely. HPLC-based analysis i s equi es he con e sion o Moco/MPT in o he
s able luo escen de i a i e Fo mA [20] and o MPT-AMP in o Fo mA-AMP [11,12], espec i ely. The e o e, 10–30
μl o he pooled elu ion ac ions (con aining ∼100 pmol p o ein) we e added o p e-mixed oxida ion p epa a ions
(each con aining 800 μl 0.1 M T is-HCl, pH 7.2 + 100 μlacidi ied1%I
2/2%KIsolu ion).TheI
2/KI s ock-solu ion
was p epa ed as desc ibed [22] and di ec ly be o e i s use o Moco/MPT oxida ion, HCl was added ( inal concen-
a ion =∼1 M). The concen a ion o he p o ein solu ion was de e mined using he B ad o d assay (Ro i-Quan ;
Ro h) wi h bo ine se um albumin se ing as a concen a ion s anda d [14]. Oxida ion o he p o eins was pe o med
o e nigh o a leas 16 h a 22◦C. A e oxida ion, p ecipi an s we e emo ed by a cen i uga ion s ep (16,000 ×g,
10 min, oom empe a u e). Nex , wice 450 μl o he supe na an was ans e ed in o esh eac ion ubes yielding
sample 1 and 2, espec i ely. Residual iodine was educed by he addi ion o 50 μlo an1%w/ aqueousasco bic
acid solu ion o bo h samples. Subsequen ly, 200 μlo a1MT issolu ionand13μl 1 M MgCl2we e added o each
sample. Fo quan i ica ion o Fo mA, 1 U o alkaline phospha ase (New England Biolabs) was added o sample 1. Fo
quan i ica ion o Fo mA-AMP, 1 U o phosphodies e ase I (MP Biomedicals) was added o sample 2. Bo h samples
we e incuba ed a oom- empe a u e o a leas 16 h. A e wa d, he alkaline phospha ase ea ed sample 1 was eady
o analysis. To sample 2 (p ocessed wi h phosphodies e ase I), 1 U alkaline phospha ase was added o con e Fo mA
– esul ing om Fo mA-AMP deadenyla ion – in o dephospho Fo mA sui able o HPLC-based Fo mA quan i ica-
ion. A e ano he o e nigh incuba ion s ep (a leas 16 h) a oom empe a u e, sample 2 was also eady o analysis
(Figu e 1 illus a es he p o ocol desc ibed he e). HPLC analysis was ca ied ou a oom empe a u e using a e e sed
phase C-18 column (250 mm ×4.6 mm, 5 μm, Rep oSil-Pu Basic C-18 HD) and an Agilen 1100 sys em consis ing
o a bina y pump, au osample and luo escence de ec o . Dephospho Fo mA was elu ed a a low a e o 1 ml min−1
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Figu e 1. P o ocol o Fo mA based Moco/MPT and MPT-AMP quan ifica ion
Fo mA-based Moco/MPT and MPT-AMP quan i ica ion in ol es six s eps. I easible he p o ein p epa a ion (s ep I) should be
ca ied ou unde low oxygen condi ions o p e en oxida i e damage o Moco/MPT and/o MPT-AMP. Di ec ly upon p epa a ion, an
aliquo o he ecombinan p o ein is used o Moco/MPT and/o MPT-AMP quan i ica ion ia Fo mA (s ep II). Oxida ion is achie ed
by adding acidi ied I2/KI solu ion o he p o ein sample. In s ep III, he oxidized p o ein sample (con aining Fo mA and Fo mA-AMP) is
spli . Excess iodine is educed wi h asco bic acid and he pH o he samples is adjus ed o basic condi ions sui able o subsequen
enzyma ic de-adenyla ion (phosphodies e ase I, PDI) o enzyma ic dephospho yla ion (alkaline phospha ase, AP). A e o e nigh
(ON) incuba ion, dephospho Fo mA de i ed om enzyma ic dephospho yla ion can be di ec ly analyzed ia HPLC, while Fo mA
de i ed om he Fo mA-AMP de-adenyla ion (sample 2) is subjec ed o a dephospho yla ion s ep by AP. Fo mA amoun s de i ed
om Moco/MPT (sample 1) o de i ed om bo h MPT-AMP and Moco/MPT (sample 2) a e quan i ied. To de e mine he MPT-AMP
amoun wi hin he sample, Fo mA quan i ied in sample 1 needs o be sub ac ed om Fo mA quan i ied in sample 2. Please no e
ha a ia ions in he Fo mA amoun s quan i ied may be ela ed o he e ec i eness o Moco/MPT and/o MPT-AMP ex ac ion om
i s p o ein en i onmen du ing he oxida ion p ocedu e.
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using an isoc a ic mobile phase con aining 5 mM ammonium ace a e and 15% ( / ) me hanol, and had a speci ic
e en ion ime o 5.25 min. Dephospho Fo mA was de ec ed luo ome ically (λex =302 nm, λem =451 nm). All
da a we e collec ed and p ocessed wi h OpenLab CDS Ve sion 2.2.0.600. Calib a ion was ca ied ou using syn he ic
dephospho Fo mA [21] as calib a ion s anda d. Fo se ial dilu ion o syn he ic dephospho Fo mA, he p o ein- ee
Fo mA p epa a ion-bu e (see abo e) was used.
Cnx1E MPT-AMP hyd olysis assay
The Cnx1E MPT-AMP hyd olysis assay was essen ially pe o med as desc ibed ea lie [14]. MPT-AMP and
Moco/MPT we e quan i ied as desc ibed abo e.
Induc i ely coupled plasma mass spec ome y
Quan i ica ion o he Cnx1E molybdenum con en was ca ied ou using an Agilen 7700 Se ies induc i ely coupled
plasma mass spec ome y (ICP-MS; Agilen Technologies). Fo calib a ion, a s anda d cu e o sodium molybda e
(1–20 mg/l, Fluka) was used. P o ein solu ions and s anda ds we e mixed au oma ically using hodium (Rh(NO3)3)
as an in e nal s anda d. All alues we e co ec ed o he molybdenum backg ound o con ol samples (bu e wi hou
p o ein). Da a collec ion and p ocessing we e ca ied ou using he MassHun e wo k s a ion so wa e.
C ys alliza ion, da a collec ion and model building
P io o c ys alliza ion, he p o ein was concen a ed o abou 30 g/l using Vi aspin concen a o columns wi h a
molecula weigh cu -o o 30 kDa. Concen a ed p o ein solu ions we e supplemen ed wi h 0.015 M o bo h MgCl2
and ADP. Bes c ys als o Cnx1E a ian D274E we e ob ained om a ious condi ions o he Mo pheus sc een
(Molecula dimensions). The c ys als we e lash-cooled in liquid ni ogen and subjec ed o X- ay di ac ion expe -
imen s on beamline P11, ope a ed by DESY a he PETRA III synch o on (Hambu g, Ge many) [23]. The da a se
was p ocessed wi h au oPROC [24] and co ec ed o aniso opy wi h STARANISO [25]. The c ys allog aphic phase
p oblem was sol ed wi h Phase [26] by ansplan ing phases om he p e iously de e mined Cnx1E wild- ype s uc-
u e (PDB en y: 6ETD, [15]). The ini ial s uc u e was imp o ed by e inemen wi h Bus e 2.10.3 [27] and ebuilding
in Coo [28]. Amino acids wi h diso de ed side chains we e modeled as s ubs ex ending only o he β-ca bon a om.
In con as , amino acids wi h a diso de ed backbone we e no a all included in he s uc u al model and a e e lec ed
by chain b eaks. To a oid clashes be ween c ys allog aphic neighbo s nea a special posi ion, D435 and I436 we e
modeled as s ubs despi e in e p e able side chain densi y. Du ing he e inemen , he a omic displacemen ac o s
we e ea ed as being iso opic and domain displacemen was accoun ed o by modeling wo igid body domains un-
de going ansla ion/lib a ion/sc ew ib a ional mo ion. The e inemen was s opped a e Rwo k and R ee con e ged.
The iles con aining he s uc u e ac o s and he s uc u al model we e deposi ed wi h he P o ein Da a Bank wi h
accession numbe 6RMS. The comple e da a collec ion and e inemen s a is ics a e shown in Table 1.
Resul s
Cnx1E ca alyzed MPT-AMP hyd olysis
We ecen ly epo ed a high- esolu ion Cnx1E s uc u e in complex wi h ac i e si e bound Mg2+-AMP and molyb-
da e [15]. Howe e , he co-c ys alliza ion o he Cnx1E enzyme subs a e complex was no possible, hus exclud-
ing he s uc u e assis ed elucida ion o Cnx1E eac i i y. P e iously, he in i o ans e o MPT-AMP on he e-
combinan Cnx1E wild- ype enzyme was epo ed [13,14]. Using hese me hods, he ou ine p oduc ion o Cnx1E
wi h MPT-AMP occupancies su icien o co-c ys alliza ion expe imen s was no success ul, which could be due
o Cnx1E- MPT-AMP hyd olysis ac i i y e ained in he c ys alliza ion condi ions. We iden i ied Cnx1E ac i e si e
esidues Th 198, Glu201, Asp242 and Asp274 as po en ial a ge s o si e-di ec ed mu agenesis o abolish MPT-AMP
hyd olysis ac i i y. These esidues a e conse ed among Mo-inse ases om a ious species (Figu e 2C) and we e
shown o in e ac wi h he Mg2+ ion wa e shell molecules (Figu e 2A, [15]) in he p e iously published Cnx1E s uc-
u e [15]. They appea o be c ucial o he p ope posi ioning o he Mg2+ ionwi hin heac i esi eandmayplay
a ole in he ac i a ion o a wa e molecule o he nucleophilic a ack o he MPT-AMP phospha e-phospha e bond
[13]. In Aspe gillus nidulans, heexchangeo heA abidopsis halianaAsp274 co esponding esidue (Asp522) o
a glu ama e esidue esul s in a comple e loss o Mo-inse ase unc ionali y [29]. We p oceeded o cha ac e ize he
co esponding Cnx1E exchange a ian (D274E).
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Figu e 2. Cnx1E magnesium coo dina ing esidues
(A)Mg
2+-AMP in e ac ing amino acid esidues a e shown in simple s ick ep esen a ion and numbe ed. P o ein o Mg2+-AMP in-
e ac ions o Cnx1E a ian D274E a e iden ical wi h he excep ion o he missing in e ac ion o esidue E274. The a oms o esidue
D274 we e shown semi- anspa en o illus a e his. The Cnx1E bound AMP molecule is shown in ball-and-s ick ep esen a ion.
The single co-c ys allized magnesium ion is shown as g een sphe e, coo dina ed wa e molecules as g ay sphe es. Hyd ogen
bonds a e ep esen ed by dashed, g ay lines. I disce nible, a owheads poin o he hyd ogen bond accep o wi h co esponding
dis ances gi en in ˚
Angs ¨
oms ( ˚
A). (B) Schema ic ep esen a ion o he A abidopsis haliana Cnx1 enzyme domain s uc u e [14,15].
The i s and las esidues o he domains a e indica ed. Fo Cnx1E subdomains I o IV a e indica ed. Residues in ol ed in di ec ed
Mg2+ in e ac ions a e gi en abo e he Cnx1E domain s uc u e. (C) Pa ial sequence compa ison o A abidopsis haliana (A ), Ra us
no egicus (Rn), Aspe gillus nidulans (An) and Esche ichia coli (Ec) Cnx1E homologs. Cnx1 esidues in ol ed in di ec ed Mg2+ in e -
ac ions a e indica ed by whi e iangles wi h he co esponding Cnx1 amino acid posi ions gi en abo e. S ic ly conse ed esidues
a e highligh ed in black, conse ed esidues a e highligh ed in g ay. The alignmen was gene a ed wi h Clus al Omega. Figu e pa
B and he co esponding cap ion was o iginally published in [15] (h ps://po landp ess.com/biochemj/a icle/475/10/1739/49736/)
and has been modi ied he e.
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Figu e 3. Biochemical cha ac e iza ion o CNX1E a ian D274E
(A) Recombinan Cnx1E wild- ype (w ) p o ein and a ian D274E we e analyzed o co-pu i ied molybdenum co ac o (Moco) /
molybdop e in (MPT), Mo and adenyla ed MPT (MPT-AMP) a e exp ession and pu i ica ion om E. coli s ain RK5206 [30]. Analysis
o he ecombinan p o ein was ca ied ou as desc ibed in he ‘Ma e ials and Me hods’ sec ion. Ba s ep esen he s anda d
de ia ion, esul ing om h ee ull eplicas. (B) HPLC elu ion p o ile o 5.258 pmol syn he ic dephospho Fo mA. A ep esen a i e
calib a ion cu e used o quan i a i e analysis shown in panel (B) is shown as a second inse benea h he chemical s uc u e
o Fo mA (Y =PO3H−). Fo dephospho Fo mA Y =H, [21]. The lowe de ec ion limi was 0.328 pmol dephospho Fo mA. (C)
P oposed mechanism o he con e sion o Moco (1) o Fo m A (5). Fo ma ion o he iple bond o Fo m A equi es p io opening
o he dihyd opy an ing p esen in Moco (1), because o he wise high s ain ene gy would a ise. Thus, as he i s s ep, acidic
hyd olysis o he N,O-ace al moie y o Moco mus happen i s , possibly a o ding bicyclic dihyd op e in 2. Fo mally, wo equi alen s
o molecula iodine a e equi ed o oxidize 1 o Fo m A (5). Gi en he high a ini y o iodine o sul u , i is p oposed ha one o
he wo sul u a oms is iodina ed elec ophilically, as i has been obse ed o molybdenum sul u complexes [34]. The libe a ed,
nucleophilic iodide could a ack he second sul u a om (s uc u e 3). Elimina ion o , p obably uns able, diiodina ed di hiomolybda e
would gene a e he alkyne moie y o Fo m A. In he inal s ep, a second equi alen o iodine may be consumed o he oxida ion
o dihyd op e in 4 o p e in 5. Th oughou he sequence, Mo(VI) would ha e kep i s oxida ion s a e. Oxida i e desul u a ion and
dehyd ogena ion o he p e in uni migh also occu in he e e sed o de . When s a ing om MPT, which lacks molybdenum, a
simila pa hway would lead o he o ma ion o wo molecules o HSI, which would be equilib ium wi h o he iodina ed sul u species
[35,36]. Nex o Fo mA also Fo mB was desc ibed as s able, luo escen Moco/MPT de i a i e [22]. In he absence o KI3, boiling
o Moco-con aining enzymes a pH 7 in he p esence o 0.01 M T is-HCl has led p edominan ly o Fo m B (11, Supplemen a y
Figu e S3). Fo ma ion o he hiophene ing o 11 equi es o a ion a ound he double bond, p esumably ia he hione au ome
6 ha is o med a e hyd olysis o he Mo-S-bond and au ome iza ion. A i s p oposed mechanism o i s o ma ion is gi en in
Supplemen a y Figu e S3.
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Table 1 Da a collec ion and efinemen s a is ics
Cnx1E-D274E
Wa eleng h ( ˚
A) 0.9537
Space g oup I222
Uni cell pa ame e s
a(˚
A) 64.84 +
−0.04
b(˚
A) 119.48 +
−0.06
c(˚
A) 137.87 +
−0.06
αࣕβࣕγ(◦) :=90
Resolu ion ( ˚
A)
dhkl,max –dhkl,min 90.01–1.74 (1.81–1.74)
dh00,e 1.74
d0k0,e 2.53
d00l,e 1.85
de ,mean1[dop ]1.99 [∼1.8]
No. o eflec ions
To al 457,830 (2195)
Unique 34,684 (166)
Comple eness
Sphe ical 0.667 (0.160)
Ellipsoidal20.927 (0.654)
Mul iplici y 13.2 (13.03)
Mean I/σ(I) 25.6 (1.5)
Wilson B ( ˚
A2) 31.5
Rme ge 0.058 (1.663)
Rmeas 0.060 (1.731)
Rpim 0.016 (0.475)
CC1/2 1.000 (0.639)
No. o eflec ions used 34,673 (165)
Rwo k /R ee 0.1983 / 0.2248
No. o non-hyd ogen a oms
To al 3217
in p o ein 2974
in ligands 15
in o de ed sol en 228
A omic B- ac o s ( ˚
A2)
A e age 43.4
P o ein/Ligands/Sol en 43.3 / 61.4 / 43.3
No. o amino acid esidues
o al / o de ed 470 / 397
RMSD om ideal
bonds ( ˚
A) 0.014
angles (◦)1.65
Ramachand an (%)
a o ed 97.92
allowed 1.82
ou lie s 0.26
Numbe s in pa en heses accoun o he shell o highes esolu ion. 1E ec i e (de ) and co esponding op ical (dop ) esolu ion o he da ase de e mined
wi h EFRESOL [33]. 2Da a comple eness o a olume in ecip ocal space bounded by an ellipsoid cen e ed on {000}and wi h he dimensions a=
1/dh00,min,b=1/d0k0,min,c=1/d00l,min.
Quan i a i e analysis o Cnx1E a ian D274E
To gi e insigh s in o he impac o esidue Asp274 o MPT-AMP hyd olysis, we ini ially exp essed and pu i ied
Cnx1E a ian D274E om E. coli s ain RK5206 [30]. Wild- ype Cnx1E om RK5206 [14] se ed as a e e ence in
he subsequen ly ca ied ou biochemical compa ison. P o ein pu i ies o wild- ype Cnx1E and Cnx1E a ian D274E
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Figu e 4. Moco syn hesis ac i i ies o wild- ype Cnx1E and Cnx1E a ian D274E
(Uppe pa ) Moco o ma ion o Cnx1E wild- ype (w ) and a ian D274E (see he ‘Ma e ials and Me hods’ sec ion o de ails). Fo
he sake o compa ison, he Moco/MPT amoun s we e no malized o 100% (Cnx1E w =45.26 +/- 2.64 pmol; Cnx1E a ian D274E
=92.07 +/- 13.45 pmol). Ba s ep esen he s anda d de ia ion, esul ing om i e ull eplicas. The linea eg ession i o bo h
eac ions is shown. Lowe pa : Wild- ype Cnx1E ca alyzed Moco o ma ion occu s wi h 3.28 +/- 0.31 pmol Moco pe min, whe eas
Moco/MPT deg ada ion was obse ed o Cnx1E a ian D274E (1.28 +/- 0.37 pmol Moco/MPT pe min).
we e ound o be equi alen (Supplemen a y Figu e S1), hus allowing hei di ec compa ison wi h espec o molyb-
da e / MPT-AMP binding and hyd olysis ac i i y. Fo Moco, me aboli e quan i ica ion syn he ic dephospho Fo mA
[21] (Figu e 3B) may be used as calib a ion s anda d o HPLC-based Fo mA quan i ica ion, ep esen ing a sui able
al e na i e o he calib a ion me hod desc ibed ea lie [19,20]. Ha ing hands on syn he ic dephospho Fo mA o he
HPLC calib a ion likewise allowed us o es ablish as e Fo mA sample p epa a ion p o ocols (Figu es 1 and 3C, see
‘Ma e ials and Me hods’ sec ion o de ails). Fo mA based HPLC analysis e ealed Cnx1E a ian D274E o accumu-
la e MPT-AMP (0.17 +/- 0.03 MPT-AMP pe monome ), while wild- ype Cnx1E was ound o be co-pu i ied wi h
signi ican ly less MPT-AMP (0.06 +/- 0.02 molecules pe monome , Figu e 3A). Nex o MPT-AMP also Moco/MPT
was ound o be co-pu i ied wi h bo h p o eins (D274E =0.05 +/- 0.01 molecules pe monome , wild- ype Cnx1E =
0.08 +/- 0.03 molecules pe monome , Figu e 3A). Since he quan i ied Mo-amoun was 0.27 +/- 0.05 (D274E, Figu e
3A), we p esume ha exclusi ely Moco bu no MPT was co-pu i ied wi h Cnx1E a ian D274E. Since MPT-AMP
and molybda e a e bound equimola by Cnx1E [13], we deduce ha nex o Mo bound in Moco, ex an Mo quan i ied
by ICP-MS o igina es om enzyme bound molybda e awai ing hyd olysis d i en Mo-inse ion in o MPT-AMP. Fo
wild- ype Cnx1E, he o al Mo-amoun quan i ied was 0.12 +/- 0.01 (Figu e 3A) sugges ing ha nex o Moco and
MPT-AMP + molybda e, mino amoun s o MPT we e co-pu i ied he e.
In i o Moco syn hesis
Nex o he quan i ica ion o enzyme bound MPT-AMP and molybda e, we es ed Cnx1E a ian D274E o i s abil-
i y o hyd olyze enzyme bound MPT-AMP. The subs a e bound enzymes analyzed we e ob ained upon in i o
MPT-AMP loading [14]. A ully de ined in i o sys em was hen employed o documen he impai ed MPT-AMP
hyd olysis capaci y o Cnx1E a ian D274E (Figu e 4). To do so, p io o he expe imen , he Cnx1E wild- ype and
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Figu e 5. Close-up on he Cnx1E ac i e si e
(A) Wild- ype Cnx1E wi h Asp274 in e ac ing wi h he wa e shell o Mg2+.(B) Cnx1E a ian D274E wi h he diso de ed loop egion
indica ed by a b oken line. Residue 274 could no be loca ed in he elec on densi y due o diso de .
D274E concen a ions we e adjus ed o ∼16% MPT-AMP sa u a ion. Cnx1E a ian D274E shows no Moco syn he-
sis capaci y (Figu e 4). Howe e , we documen ed Moco/MPT deg ada ion he e (-1.28 +/- 0.37 pmol Moco/MPT pe
min) ha we a ibu e o oxida i e damage o Moco/MPT. This also explains MPT-AMP consump ion (Figu e 4) o
he demons a i ely inac i e ( his wo k and [29]) Cnx1E a ian D274E. To shed ligh on he molecula unc ion o
Cnx1E esidue Asp274 wi h espec o magnesium ion coo dina ion (Figu e 2A), we p oceeded o sol e he s uc u e
o he Cnx1E D274E a ian .
The s uc u e o Cnx1E a ian D274E
The o e all s uc u e o Cnx1E a ian D274E esembles he Cnx1E wild- ype s uc u e published ecen ly [15]. As
a no able di e ence, no in e p e able elec on densi y is isible in he a ian s uc u e o he egion comp ising
amino acids 269 h ough 274. Howe e , he ac i e si e magnesium ion [15] was ound o be in place (Figu e 5). The
diso de ed egion 269 o 274 was con i med o be highly dynamic in D274E h ough ensemble e inemen [31]. In he
wild- ype s uc u e, esidue Asp274 in e ac s wi h he oc ahed al wa e shell o he ca aly ically ele an magnesium
ion and is he e o e in ol ed in i s coo dina ion (Figu es 2A and 5A). Replacemen o Asp274 wi h glu ama e seems
o p e en his in e ac ion and o gi e way o eo ien a ion and diso de o ha egion (Figu e 5B).
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