S uc u al Elucida ion and Enginee ing o a Bac e ial Ca bohyd a e
Oxidase
Alessand o Bo e io, Wahyu S. Widodo, La s L. San ema, Hen ië e J. Rozeboom, Rui e Xiang,
Víc o Gualla , And ea Ma e i, and Ma co W. F aaije*
Ci e This: Biochemis y 2023, 62, 429−436
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ABSTRACT: Fla in-dependen ca bohyd a e oxidases a e al-
uable ools in bio echnological applica ions due o hei high
selec i i y in he oxida ion o ca bohyd a es. In his s udy, we
epo he biochemical and s uc u al cha ac e iza ion o a ecen ly
disco e ed ca bohyd a e oxidase om he bac e ium Rals onia
solanacea um, which is a membe o he anillyl alcohol oxidase
la op o ein amily. Due o i s excep ionally high ac i i y owa d N-
ace yl-D-galac osamine and N-ace yl-D-glucosamine, he enzyme
was named N-ace yl-glucosamine oxidase (NagOx). In con as o mos known ( ungal) ca bohyd a e oxidases, NagOx could be
o e exp essed in a bac e ial hos , which acili a ed de ailed biochemical and enzyme enginee ing s udies. S eady s a e kine ic analyses
e ealed ha non-ace yla ed hexoses we e also accep ed as subs a es albei wi h lowe e iciency. Upon de e mina ion o he c ys al
s uc u e, s uc u al insigh s in o NagOx we e ob ained. A la ge ca i y con aining a bico alen ly bound FAD, e he ed ia his idyl and
cys einyl linkages, was obse ed. Subs a e docking highligh ed how a single esidue (Leu251) plays a key ole in he accommoda ion
o N-ace yla ed suga s in he ac i e si e. Upon eplacemen o Leu251 (L251R mu an ), an enzyme a ian was gene a ed wi h a
d as ically modi ied subs a e accep ance p o ile, uned owa d non-N-ace yla ed monosaccha ides and disaccha ides. Fu he mo e,
he ac i i y owa d bulkie subs a es such as he isaccha ide mal o iose was in oduced by his mu a ion. Due o i s ad an age o
being o e exp essed in a bac e ial hos , NagOx can be conside ed a p omising al e na i e enginee able bioca alys o selec i e
oxida ion o monosaccha ides and oligosaccha ides.
Fla in-dependen ca bohyd a e oxidases make up a g owing
class o enzymes ha can be subdi ided in o wo majo
la op o ein amilies depending on hei s uc u al old: he
glucose-me hanol-choline (GMC) amily and he anillyl
alcohol oxidase (VAO) amily.
1−4
Fla op o ein oxidases ac ing
on ca bohyd a es ep esen highly aluable ools in bio-
echnology due o hei abili y o be highly egioselec i e and
e icien in he oxida ion o suga s. The mos used
ca bohyd a e oxidase is glucose oxidase (EC 1.1.3.4)
5
om
Aspe gillus nige , which ca alyzes he con e sion o β-D-glucose
in o D-glucono-1,5-lac one (C1 oxida ion) and belongs o he
GMC amily. Ano he known GMC- ype ca bohyd a e oxidase
is py anose oxidase, which is also abundan ly p esen in ungal
p o eomes and ac s on monosaccha ides such as D-glucose bu
ypically oxidizes he C2 hyd oxy g oup o hexoses.
6,7
VAO-
ype ca bohyd a e oxidases, ins ead, a e known o be p ima ily
ac i e owa d oligosaccha ides.
8
The i s example was
desc ibed mo e han 30 yea s ago, glucooligosaccha ide
oxidase (GOOX)
9
om Ac emonium s ic um. In he pas
se e al yea s, se e al mo e enzymes belonging o he same
amily ha e been disco e ed and cha ac e ized, mainly o a
ungal o igin: chi ooligosaccha ide oxidase (Chi O)
10
om
Fusa ium g aminea um, xylooligosaccha ide oxidase (XylO)
11
om Mycelioph ho a he mophila, and lac ose oxidase (LaO)
12
om Mic odochium ni ale. Recen ly, an enzyme wi h analogous
ea u es has been iden i ied in plan s.
13
F om a s uc u al poin
o iew, VAO-like oxidases sha e a common old ha is
undamen ally di e en om ha o GMC- ype enzymes.
14
The s uc u e can be di ided in o wo majo domains: a
subs a e binding domain (S-domain) o med by he C-
e minal pa o he p o ein sequence and a la in binding
domain (F-domain) a he N- e minus. In gene al, he ac i e
si e o VAO-like oxidases has a la ge binding g oo e compa ed
o ha displayed by he GMC-oxidases,
15
explaining hei
b oade subs a e accep ance p o iles. Fu he mo e, enzyme
enginee ing s udies
16
showed how he subs a e speci ici y o
hese enzymes is s ic ly dependen on he esidues ha a e
p esen in he S-domain. The applica ions o ca bohyd a e
oxidases a e mul i old. Known examples can be ound in ood
applica ions
17
and biosenso s.
18
He e, we epo on he
biochemical cha ac e iza ion, s uc u al elucida ion, and
s uc u e-inspi ed enginee ing o a ecen ly disco e ed
Special Issue: P o ein Enginee ing
Recei ed: May 27, 2022
Re ised: July 11, 2022
Published: July 26, 2022
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bac e ial VAO-like ca bohyd a e oxidase.
19
Wi h he de eloped
exp ession sys em, elucida ed c ys al s uc u e, and es ablished
biochemical ea u es (subs a e ange, kine ics, and edox
po en ial), his newly disco e ed ca bohyd a e oxidase
ep esen s a p omising bioca alys ha can be uned o
speci ic applica ions.
■MATERIALS AND METHODS
Chemicals. Ni-Sepha ose 6 Fas Flow was om Cy i a, and
N,N′-diace ylchi obiose was om To on o Resea ch Chem-
icals. All o he chemicals we e o de ed om Sigma-Ald ich.
Cloning, T ans o ma ion, Mu agenesis, and Exp es-
sion. A syn he ic gene encoding NagOx, codon-op imized o
Esche ichia coli wi h BSAI si es a he 5′and 3′ e mini (Twis
Bioscience), was cloned wi h he Golden Ga e me hodology in
a pBAD His-SUMO ec o . Fo ans o ma ion, 2 μL o
plasmid was added o 50 μL o NEB10βRbCl compe en cells
and incuba ed on ice o 30 min. Cells we e hen hea shocked
a 42 °C o 40 s and incuba ed again on ice o 2 min. Then,
250 μL o p ewa med LB-SOC medium was added, and he
cells we e incuba ed o 1 h a 37 °C; 50 μL o he eco e ed
cells was pla ed on LB-aga supplemen ed wi h 50 μg mL−1
ampicillin and incuba ed o e nigh a 37 °C. Plasmid isola ion
was pe o med, and cloning was e i ied h ough sequencing. A
p einoculum o 5 mL o LB-amp (50 μg.mL−1) was g own
o e nigh a 37 °C and used o inocula e 2 L ba led lasks
con aining 400 mL o Te i ic B o h medium supplemen ed
wi h 50 μg mL−1ampicillin. Flasks we e incuba ed a 37 °C
un il an OD600 o 0.6−0.8 was eached. Exp ession was
induced wi h 0.02% L-a abinose, and cul u es we e le a 24 °C
o 24 h be o e being ha es ed. Cells we e ha es ed by
cen i uga ion (6000 pm, 20 min, 4 °C) and lash- ozen in
liquid ni ogen.
To p epa e enzyme mu an s, p ime s we e o de ed om
Eu o ins genomics. All o he mu a ions we e ca ied ou using
he QuickChange me hodology.
20
The PCR mix (25 μL)
consis ed o he ollowing componen s: P uUl a II Ho s a
PCR Mas e Mix (12.5 μL), 1 μL o p ime w (10 μM), 1 μL
o p ime e (10 μM), 1 μL o plasmid (100 ng/μL), 0.4 μL
o DMSO, and MQ wa e up o 25 μL.
P o ein Pu i ica ion. Cell pelle s we e esuspended in
bu e A [100 mM KPiand 500 mM NaCl (pH 7.5)] wi h a
3:1 olume (millili e s):mass (g ams) a io. Then, 0.10 mM
PMSF and 1.0 mM β-me cap oe hanol we e added o he lysis
solu ion o p e en p o ein deg ada ion. Cells we e dis up ed
by sonica ion (5 s on, 5 s o , 70% ampli ude o a o al o 10
min) and hen cen i uged a 11 000 pm o 1 h. The esul ing
supe na an was loaded on a g a i y column con aining 3 mL
o Ni Sepha ose p e iously equilib a ed wi h bu e A.
A e a washing s ep [3 column olumes (CV)] wi h bu e
B [50 mM KPi, 500 mM NaCl, and 20 mM imidazole (pH
7.5)], he p o ein was elu ed (2 CV) wi h bu e C [50 mM
KPi, 500 mM NaCl, and 500 mM imidazole (pH 7.5)]. Elu ion
bu e was hen exchanged agains s o age bu e [50 mM
po assium phospha e bu e (pH 6.5) and 100 mM NaCl]. The
concen a ion o he pu i ied enzyme was measu ed using he
ex inc ion coe icien o 6-S-cys einyl-bound FMN (ε445 = 11.6
mM−1cm−1).
21,22
pH Op imum. Enzyme ac i i y was analyzed by moni o ing
oxygen consump ion a 25 °C using 1.0 μM pu i ied enzyme
[in 50 mM KPi(pH 6.5)] wi h 5.0 mM D-glucose. The o al
eac ion olume was 1.0 mL. Oxygen consump ion was
measu ed using an Oxyg aph Plus sys em (Hansa ech
Ins umen s L d.), and he eac ion was ini ia ed by adding
he enzyme. Ini ial a es we e de e mined om he ini ial linea
pa s o he eac ion cu es.
The mal S abili y. Due o he bico alen p o ein−FAD
linkage, la in luo escence is quenched, and hus, he
The moFAD me hod could no be used o moni o enzyme
un olding.
22
The e o e, he he mos abili y o he enzyme was
de e mined using he The mo luo assay.
23
Fo hese he mal
un olding assays, ∼150 μM enzyme [50 mM KPi(pH 6.5)]
was dilu ed 10- old in a ious es ed bu e s (in duplica e) a
di e en pH alues mixed wi h SYPRO o ange dye.
24
The
assay was pe o med using a RT-PCR he mocycle (CFX96
om Bio-Rad). Measu emen s s a ed a 20 °C, and he
empe a u e was inc eased a a a e o 1 °C/min un il 95 °C.
S eady S a e Kine ics. Oxidase ac i i ies on he es ed
ca bohyd a es we e moni o ed on he basis o he o med
hyd ogen pe oxide. Hyd ogen pe oxide o ma ion was coupled
o he ac i i y o ho se adish pe oxidase (HRP) and
ch omogenic pe oxidase subs a es (AAP and DCHBS).
25
Abso bance measu emen s we e conduc ed on a JASCO V-660
ins umen a 515 nm (ε515 = 26 mM−1cm−1). Ra es a
di e en subs a e concen a ions we e p ocessed in G aphPad
P ism and i ed using a egula Michaelis−Men en o mula
esul ing in KM(millimola ) and kca (in e se seconds) alues.
P o ein C ys alliza ion, S uc u al Elucida ion, and
Docking. A e clea age wi h SUMO p o ease, pu i ied
p o ein was loaded on a Supe dex200 10/300 column (Cy i a)
using an AKTA pu i ie . Two wa eleng hs (280 and 447 nm)
o moni o ing p o ein elu ion we e used du ing he
pu i ica ion. The cen al ac ions o he peak we e pooled
oge he and concen a ed un il a concen a ion o 12.5 mg/
mL was eached on he basis o he la in abso p ion peak.
Di e en c ys alliza ion condi ions we e es ed using a
Mosqui o c ys alliza ion obo (TTP LabTech, Melbou n,
U.K.). La ge homboid-shaped yellow c ys als appea ed unde
di e en condi ions. A e op imiza ion, he bes condi ion was
ob ained h ough si ing d op apo di usion using 19%
PEG3350 and 0.19 M sodium ni a e; 25% glyce ol was used
as a c yop o ec an , and c ys als we e lash- ozen in liquid
ni ogen and sen o ESRF o da a collec ion. The bes c ys al
di ac ed a 1.5 Å esolu ion using he MASSIF-1
26
beamline.
Da a we e scaled using XDS. Molecula eplacemen was done
using Phenix.
27
S uc u al e inemen was done using COOT
28
and REFMAC5
29
o he CCP4 package.
29
The de ailed
s a is ics o he collec ed da a se a e summa ized in Table 1.
Docking simula ions we e pe o med using he high-
esolu ion c ys al s uc u e o NagOx. Yasa a
30
was used as a
ool applying he AMBER IPQ o ce ield wi h a cell o 20 Å ×
20 Å ×20 Å, which included he whole ac i e si e. The
expe imen was conduc ed wi h 100 uns using Au oDock
Vina.
31
The esul s ha showed a p ope con o ma ion
unde wen ene gy minimiza ion in Yasa a. The de aul se ings
we e used o he compu a ional ools.
Redox Po en ial De e mina ion. The edox po en ial o
NagOx was de e mined using he xan hine/xan hine oxidase
me hodology.
32,33
The eac ion was pe o med in 50 mM KPi
bu e (pH 7.5) a 25 °C using a 1 mL qua z cu e e. The
eac ion mix u e consis ed o 5.0 μM benzyl iologen, 5.0 μg/
mL ca alase, 400 μM xan hine, a ca aly ic amoun o xan hine
oxidase, 0.5 μM 5-hyd oxyme hyl u u al oxidase (HMFO),
and 20 mM HMF. An anae obic condi ion was c ea ed by
lushing he cu e e wi h a gon o 15 min a e which he
HMF/HMFO sys em assu ed ully anoxic condi ions.
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430
Xan hine was added o ini ia e he edox i a ion. Spec a we e
eco ded o 1 h. Me hylene blue (E0= 11 mV) was ound o a
sui able dye o de e mining he edox po en ial. The EM alue
was calcula ed by applying he Ne ns equa ion.
32,33
Subs a e Induced-Fi Simula ions. Subs a e induced-
i simula ions we e pe o med wi h PELE (P o ein Ene gy
Landscape Explo a ion), a so wa e ha combines Mon e
Ca lo (MC) sampling wi h p o ein s uc u e p edic ion
algo i hms.
34
B ie ly, a each MC simula ion s ep, i execu es
(i) a pe u ba ion phase, including a andom ansla ion and
o a ion o he ligand and a no mal mode displacemen o he
enzyme backbone, and (ii) a elaxa ion phase, comp ising a
side chain packing op imiza ion and o e all minimiza ion; new
con o ma ions a e hen accep ed o ejec ed on he basis o he
Me opolis c i e ion. The ini ial pose was p epa ed om he
newly de e mined c ys al s uc u e using he P o ein
P epa a ion Wiza d,
35
whe e he L251R mu an was ini ially
in oduced wi h he builde in Maes o.
36
D-Glucose was
downloaded om pubchem and p epa ed wi h LigP ep
37
be o e an ini ial docking using Glide.
38
■RESULTS
Cha ac e is ics and S abili y o he Enzyme. A e
success ully cloning he NagOx-enoding gene in a pBAD-His-
SUMO ec o , we o e exp essed he SUMO- used enzyme in
E. coli NEB10-β. App oxima ely 40 mg o yellow-colo ed
enzyme could be pu i ied om a 1 L cul u e using a single
IMAC pu i ica ion s ep. Sodium dodecyl sul a e−polyac yla-
mide gel elec opho esis (SDS−PAGE) analysis showed a
p o ein band a ∼70 kDa (Figu e S1). This esul ma ches well
wi h he p edic ed molecula weigh o he His-SUMO-NagOx
p o ein (72 kDa). Incuba ion o he SDS−PAGE gel wi h 5%
ace ic acid o 10 min e ealed unde ul a iole (UV) ligh he
p esence o a co alen ly bound la in (Figu e S1). The pu i ied
enzyme displayed a cha ac e is ic la in UV− isible abso p ion
spec um wi h maxima a 447 and 375 nm (Figu e 1).
The he mos abili y o NagOx was p obed in di e en
bu e s wi h a ange o pH alues om 5 o 9 using he
The mo luo me hodology (Table 2). The highes Tm alue o
50 °C was ob ained in 50 mM KPibu e (pH 6). O e a wide
pH ange, he enzyme displayed i s highes he mos abili y
unde somewha acidic condi ions. Fu he mo e, we obse ed
ha addi i es such as NaCl (≤150 mM) and glyce ol (≤5%)
did no imp o e o educe he s abili y o he enzyme. Be o e
s eady s a e kine ic analyses we e pe o med, he pH op imum
o he ac i i y o he enzyme was also de e mined (Figu e 2).
In con as o he pH op imum o s abili y, NagOx is mo e
Table 1. Da a Collec ion and Re inemen S a is ics o
NagOx
PDB en y 7ZZK
space g oup P212121
uni cell axes (Å) 87.96, 105.01, 120.67
uni cell angles (deg) 90, 90, 90
esolu ion (Å) 45.00 (1.50)
Rme ge (%) 7.1 (59.8)
Rpim (%) 5.3 (43.2)
CC1/2 0.998 (0.732)
comple eness (%) 99.7 (100)
no. o unique e lec ions 178041 (8774)
mul iplici y 2.5 (2.7)
o e all I/σ(I) 12 (2.4)
no. o p o ein esidues 972
no. o FAD molecules 2
no. o wa e molecules 1192
Wilson B- ac o (Å2) 13.9
R/R ee (%) 15.1/17.8
oo -mean-squa e de iai on o bond leng hs (Å) 0.0127
oo -mean-squa e de iai on o bond angles (deg) 1.77
Ramachand an ou lie s (%) 0.21
MolP obi y sco e 1.38 (100 h pe cen ile)
Figu e 1. Abso bance spec um o 20 μM NagOx [50 mM po assium
phospha e bu e (pH 6.5)].
Table 2. The mos abili y o NagOx
a
Tm(°C)
condi ion wild- ype NagOx Leu251A g NagOx
50 mM ci a e bu e (pH 5) 43 42
50 mM ci a e bu e (pH 5.5) 46 48
50 mM KPi(pH 6) 50 50
50 mM KPi(pH 6.5) 49 48.5
50 mM KPi(pH 7) 49 50
50 mM KPi(pH 7.5) 47.5 48
50 mM T is-HCl (pH 8) 48 49
50 mM T is-HCl (pH 8.5) 45 45.5
50 mM T is-HCl (pH 9) 44.5 45.5
a
Mel ing empe a u es we e measu ed using he The mo luo me hod
o wild- ype and L251R NagOx.
Figu e 2. pH-dependen op imum o ac i i y o NagOx. All eac ions
used 5.0 mM glucose and 1.0 μM enzyme and we e pe o med a 25
°C. Fo pH 6−8, 50 mM KPibu e was used, and o pH 8−9.5, T is-
HCl was he bu e o choice.
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Biochemis y 2023, 62, 429−436
431
ac i e a a ela i ely high pH, wi h an op imum a pH 8.5−9.
All o ou subsequen analyses we e ca ied ou in 50 mM KPi
bu e (pH 7.5).
Wi h a bico alen ly bound FAD as a co ac o , which was
expe imen ally con i med ia he elucida ion o he enzyme
s uc u e ( ide in a), NagOx may exhibi a ela i ely high
edox po en ial.
39
To e i y his, he edox po en ial o he
FAD co ac o was de e mined, using he xan hine/xan hine
oxidase me hodology. Wi h his p o ocol, a ull educ ion o
he enzyme could be obse ed wi hou any in e media e
o ma ion o a one-elec on- educed enzyme species. The
edox po en ial o NagOx was ound o be 2 mV using
me hylene blue (E0= 11 mV) as a e e ence dye (Figu e S2).
While his is a ela i ely high edox po en ial o a la op o ein,
i is in he same ange o edox po en ials p e iously epo ed
o la op o eins con aining co alen FAD ha includes a
his idyl linkage a he 8-me hyl moie y o he la in
co ac o .
40,41
Subs a e Sc eening and S eady S a e Kine ic
Analyses. To add ess he subs a e p o ile o NagOx, 37
di e en ca bohyd a es we e es ed (Table S1). A concen-
a ion o 10 mM was used, and ac i i y was assessed using he
HRP-based assay ha de ec s hyd ogen pe oxide o ma ion.
Wi h 1.0 μM NagOx, ac i i y was quickly obse ed o N-
ace yl-D-glucosamine, N-ace yl-D-galac osamine, and N,N′-
diace ylchi obiose. A e se e al minu es, pe oxide o ma ion
was also obse ed o D-glucose, D-galac ose, D-mannose,
cellobiose, and mal ose. These obse a ions indica e ha
NagOx can ac on mono- and disaccha ides and seems o ha e
a p e e ence o N-ace yla ed ca bohyd a es. On he basis o
he sc eening esul s, he s eady s a e kine ic pa ame e s we e
measu ed o he con i med ca bohyd a e subs a es men-
ioned abo e (Figu e S3 and Table 3).
The ini ial eac ion a es we e de e mined and could be
i ed success ully in all cases using he Michaelis−Men en
o mula. F om his analysis, i eme ged ha N-ace yl-D-
glucosamine and N-ace yl-D-galac osamine a e he p e e ed
subs a es o he enzyme wi h kca alues o 120−140 s−1and
s ikingly low submillimola KM alues. In e es ingly, he N-
ace yla ed disaccha ide N,N′-diace ylchi obiose was also ound
o be one o he be e subs a es by exhibi ing a lowe kca (36
s−1) and a highe KM(8.5 mM) compa ed o hose o he N-
ace yla ed monosaccha ide o m. NagOx displays simila and
ela i ely low ca aly ic e iciencies o he monosaccha ides D-
galac ose, D-glucose, and D-mannose, which was due o
ela i ely low kca (0.7−2.3 s−1) and high KM(30−92 mM)
alues. Again, a lowe ca aly ic e iciency was obse ed when
compa ed ha o he disaccha ide cellobiose wi h D-glucose.
This was mainly due o a 10- old lowe kca . These esul s
indica e ha he N-ace yl moie y plays a key ole in subs a e
ecogni ion and ha NagOx displays a be e e iciency owa d
monosaccha ides while also accep ing disaccha ides. The
subs a e accep ance p o ile is eminiscen o ha o he
ungal Chi O,
10
ye NagOx displays a be e e iciency owa d
monosaccha ides.
O e all S uc u e and Ac i e Si e o NagOx. To
unde s and he ca aly ic machine y o NagOx, we se ou o
de e mine i s h ee-dimensional s uc u e. NagOx o med la ge
homboid-shaped yellow c ys als wi h 19% PEG3350 and 0.19
M sodium ni a e ha di ac ed o 1.5 Å esolu ion. E en
hough, acco ding o size exclusion ch oma og apy expe i-
men s (Figu e S4), he enzyme showed a monome ic
con o ma ion in solu ion, wo NagOx molecules we e p esen
in he asymme ic uni (Figu e S5). The o e all s uc u e o
NagOx is simila o hose o o he known la oenzymes
belonging o he VAO amily. S uc u al alignmen wi h Chi O
[P o ein Da a Bank (PDB) en y 6Y0R] and GOOX (PDB
en y 2AXR) esul ed in oo -mean-squa e de ia ions o 1.8
and 1.9 Å, espec i ely. Inspec ion o he s uc u e e ealed
ha he isoalloxazine moie y o he FAD co ac o is
bico alen ly bound a he in e ace be ween he F-domain
and he S-domain. The F-domain comp ises esidues om he
N- e minus o esidue 205 and a small pa o he C- e minus
( esidues 456−506). On he con a y, he S-domain is
composed o esidues 206−455 (Figu e 3A). The FAD
co ac o is bico alen ly bound ia 8α-N1-his idyl and 6-S-
cys einyl linkages wi h esidues His64 and Cys123, espec i ely
(Figu e 3B). These cha ac e is ic bico alen la in−p o ein
linkages we e i s obse ed wi h GOOX (His70 and Cys130)
and a e also p esen in Chi O (His64 and Cys154).
42
The isoalloxazine ing is exposed o he sol en a ea a he
bo om pa o an open ac i e si e (Figu e 3C), while he
ibi yl and ADP moie ies o he FAD a e embedded deeply in
he F-domain. O he amino acids ha o m he ac i e si e
adjacen o he edox-ac i e isoalloxazine moie y a e a s ing o
y osines (Ty 66, Ty 137, Ty 345, Ty 414, Ty 459, and
Ty 462) wi h se e al neighbo ing pola esidues (Se 122,
Gln381, Asp383, Gln410, and Gln412). The ac i e si e sha es
many common ea u es wi h ha o sequence- ela ed
ca bohyd a e oxidases such as Chi O.
42
The same posi ion is
indeed conse ed o Ty 66, Ty 462, Gln381, Asp383, and
Gln410 (Table 4). In e es ingly, e en hough he same esidue
is conse ed o Ty 137, in NagOx he o ien a ion is owa d
he isoalloxazine ing while in Chi O (Ty 168) i is poin ing in
he opposi e di ec ion. A sho s e ch o esidues (292−313)
is no isible in he elec on densi y, which sugges s ha hey
Table 3. Appa en S eady S a e Pa ame e s o NagOx
a
wild- ype NagOx Leu251A g NagOx
subs a e K′M(mM) k′ca (s−1)k′ca /K′M(M−1s−1)K′M(mM) k′ca (s−1)k′ca /K′M(M−1s−1)
N-ace yl-D-glucosamine 0.22 ±0.04 140 ±6 64 ×10423 ±6 34 ±3 1.5 ×103
N-ace yl-D-galac osamine 0.13 ±0.02 120 ±4 93 ×1041.9 ±0.6 4.2 ±0.4 2.2 ×103
D-glucose 92 ±15 2.2 ±0.1 23 6.2 ±2.5 1.3 ±0.2 210
D-galac ose 30 ±8 2.3 ±0.02 77 7.7 ±1.8 2.1 ±0.2 270
D-mannose 36 ±5 0.71 ±0.03 20 14 ±4 5.3 ±0.5 390
N,N′-diace ylchi obiose 8.5 ±2.2 36 ±4 4 ×10352 ±4 2.7 ±0.1 52
D-cellobiose 130 ±40 0.22 ±0.03 2 80 ±26 3.2 ±0.6 40
D-mal ose >400 <0.2 0.5 22 ±4 0.7 ±0.1 34
mal o iose nd nd −210 ±30 0.34 ±0.02 1.6
a
The kine ic pa ame e s we e measu ed a 25 °C in 50 mM KPi(pH 7.5). nd indica es no ac i i y could be measu ed.
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432
o m a lexible loop wi h a disul ide b idge a i s base be ween
Cys291 and Cys316. Chi O and GOOX do no display any
lexible egion o disul ide b idge in his pa o he p o ein
s uc u e. In he ac i e si e o NagOx, a molecule o e hylene
glycol and glyce ol a e bound, p o iding a hin abou how
NagOx may in e ac wi h i s ca bohyd a e subs a e. The
e hylene glycol C1 a om is 3.0 Å abo e he N5 a om o he
la in. The O1 a om in e ac s wi h Ty 462 OH (3.1 Å) and
Gln410 NE2 (3.2 Å). This esul is in line wi h wha was
obse ed in he p e iously elucida ed s uc u es o XylO and
GOOX. In bo h cases, a Ty esidue in he analogous posi ion
was desc ibed o in e ac wi h O1 o he bound ca bohyd a e
ligand and possibly ac as a ca aly ic base. The molecule o
glyce ol is ins ead placed in a seconda y pocke . C1 is placed
3.6 Å om Leu251; O3 in e ac s wi h Asp383 OD2 (2.6 Å)
and Ty 137 OH (3.6 Å), and O2 is placed 3.4 Å om Se 122
OG.
Enginee ing NagOx owa d Ac i i y on Non-N-
ace yla ed Ca bohyd a es. Despi e se e al soaking a emp s
wi h N-ace yla ed compounds, no c ys al s uc u e in complex
wi h a ca bohyd a e was ob ained. Ins ead, an in silico analysis
was pe o med o unde s and which esidues a e in ol ed in
subs a e binding. F om ou p e ious s udy o NagOx, one
could conclude ha oxida ion occu s a C1 o NagOx
ca bohyd a e subs a es.
19
This p o ides inpu on how he
subs a e should be posi ioned wi h espec o N5 o he la in
co ac o . N-Ace yl-D-glucosamine was docked in o he
s uc u e, which e ealed a binding pose in which he N-ace yl
moie y can occupy he seconda y hyd ophobic pocke o he
ac i e si e (Figu e 4).
The obse ed binding mode o he ace yl moie y is nicely in
line wi h he bound glyce ol. The bound PEG molecule
occupies he locus in which he hexose moie y o N-ace yl-D-
glucosamine was docked in he c ys al s uc u e. The sugges ed
binding is in line wi h wha was obse ed o Chi O, whe e a
simila pocke can accommoda e N-acyl moie ies a ached a
C2.
42
The seconda y pocke in NagOx is o med by esidues
Se 122, Ty 137, Leu139, Leu251, Val334, Gln381, Asp383,
Ty 459, and Ty 462. While o Chi O, Gln268 was ound o
play a key ole in accommoda ing he N-ace yl moie y, NagOx
has a leucine (Leu251) in he analogous posi ion. The
hyd oxyl moie y a C1 o he suga poin s owa d Ty 462
(3.0 Å dis ance), which may ac as he base o igge o enable
hyd ide ans e om he subs a e o N5 o he FAD co ac o
( ide sup a). Such a mechanism is also in line wi h he dis ance
om N5 o C1 o he docked subs a e (3.4 Å).
43
The
obse ed subs a e binding mode was used as he basis o an
enzyme enginee ing e o . To al e he subs a e accep ance o
NagOx wi h espec o non-N-ace yla ed monosaccha ides
(such as D-glucose and D-galac ose) and disaccha ides (such as
cellobiose and mal ose), an enzyme a ian was gene a ed in
which Leu251 was eplaced. Because he eplacemen o
Gln268 wi h an a ginine in Chi O imp o ed he ac i i y
owa d glucooligosaccha ides,
16
we p epa ed he Leu251A g
NagOx mu an o p obe he e ec on subs a e accep ance in
his bac e ial oxidase. The Leu251A g mu an could be
o e exp essed and pu i ied wi h yields simila o hose o
wild- ype NagOx and displayed simila he mos abili y (Table
2). Nex , a s eady s a e kine ic analysis was ca ied ou (Table
3). Compa ed o ha o wild- ype NagOx, he ac i i y owa d
N-ace yl-D-glucosamine and N-ace yl-D-galac osamine was
d as ically educed. The ca aly ic e iciency dec eased ∼400-
old o bo h N-ace yla ed monosaccha ides. The ac i i y
owa d N,N′-diace ylchi obiose was also educed wi h a
dec ease in ca aly ic e iciency by 2 o de s o magni ude. The
dec eased ca aly ic pe o mance on hese subs a es was caused
by lowe kca and KM alues. These da a con i m he ole o
Leu251 in posi ioning hese N-ace yla ed ca bohyd a es in he
Figu e 3. S uc u al analysis o NagOx. (A) The F-domain is colo ed
whea , he S-domain ligh eal, and he FAD co ac o yellow. (B)
Bico alen ly bound FAD ia 8α-N1-his idyl and 6-S-cys einyl linkages
wi h a weigh ed 2Fo−Fcelec on densi y map. The con ou le el o
he map is 1.0σ. (C) Ac i e si e o NagOx. PEG and glyce ol
molecules a e colo ed g een, and he FAD co ac o is colo ed yellow.
Table 4. Compa ison o he Ac i e Si es o NagOx, Chi O,
and GOOX
a
NagOx Chi O GOOX
Ty 66 Ty 96 Ty 72
Ty 137 Ty 168 Ty 144
Ty 345 Ala341 Ala318
Ty 414 Se 410 Se 388
Ty 459 Ty 444 Ty 426
Ty 462 Ty 447 Ty 429
Se 122 Th 153 Th 129
Gln381 Gln375 Gln353
Asp383 Asp377 Asp355
Gln410 Gln406 Gln384
Gln412 Ty 408 Ty 386
a
Conse ed esidues a e shown in bold.
Figu e 4. Docked N-ace yl-D-glucosamine in he ac i e si e o NagOx.
N-Ace yl-D-glucosamine is colo ed da k g een, and he FAD co ac o
yellow. Dis ances a e exp essed in angs oms.
Biochemis y pubs.acs.o g/biochemis y A icle
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Biochemis y 2023, 62, 429−436
433
ac i e si e. In e es ingly, he ac i i y owa d non-N-ace yla ed
hexoses was imp o ed. Fo D-glucose and D-galac ose, while
he kca alues we e ha dly a ec ed, he KM alues dec eased o
alues o <10 mM, esul ing in inc eases in ca aly ic e iciency
o 9- old o D-glucose and 3.5- old o D-galac ose. In ac , he
speci ici y o he Leu251A g NagOx mu an o D-glucose (KM
= 6.2 mM) is signi ican ly highe han ha o he widely
applied and comme cially a ailable glucose oxidase om A.
nige (KM= 26 mM).
2
The ca aly ic e iciencies o D-mannose
and cellulose imp o ed 20- old. In e es ingly, he la ges
bene icial e ec on ca aly ic pe o mance was ound o he
disaccha ide mal ose (70- old imp o emen in ca aly ic
e iciency). Tes ing mal o iose e ealed a simila end.
While no signi ican ac i i y could be obse ed wi h wild-
ype NagOx, he Leu251A g mu a ion in oduced ac i i y o
his isaccha ide (KM= 210 mM, and kca = 0.34 s−1).
Modeling D-Glucose Binding in he L251R Va ian . To
unde s and he dec ease in KM o D-glucose in he L251R
a ian , we again pe o med simula ions. Mo eo e , o add ess
po en ial local con o ma ional changes, his ime we u ned o
induced- i calcula ions wi h he PELE so wa e, which is
capable o quickly mapping he con o ma ional changes
associa ed wi h p o ein−ligand in e ac ions.
44
Figu e S6a
shows he D-glucose−NagOx in e ac ion ene gy p o iles
along he (ac i e si e) induced- i simula ion o he wild
ype and he L251R mu an . Clea ly, we obse e signi ican ly
lowe in e ac ion ene gies o he enginee ed a ian a sho
ca aly ic dis ances, indica ing be e subs a e binding in e ms
o ene gies and in achie ing ca aly ic poses. Inspec ing he bes
enzyme−subs a e pose o L251R, we obse ed a good p o on
abs ac ion dis ance, 1.97 Å, which seemed o be pa ially
d i en by di ec in e ac ion wi h A g251 (Figu e S6b).
■DISCUSSION
Fla op o ein oxidases ac ing on ca bohyd a es a e e sa ile and
alued bioca alys s ypically displaying high subs a e speci ic-
i y and egioselec i i y. Ne e heless, almos all ca bohyd a e
oxidases cha ac e ized so a a e o euka yo ic o igin, and
he e o e, he exp ession in a bac e ial hos is o en challenging,
hampe ing u he enzyme enginee ing s a egies and/o
de elopmen o applica ions. The ecen iden i ica ion o a
ca bohyd a e oxidase (NagOx) om he bac e ium Rals onia
solanacea um wi h he same egioselec i i y
19
as p e iously
disco e ed ungal analogues inspi ed us o u he in es iga e
his enzyme. NagOx belongs o he VAO- ype la op o ein
oxidase supe amily and can be o e exp essed in E. coli wi h a
yield o 40 mg/L a e one a ini y ch oma og aphy pu i ica ion
s ep. We obse ed ha he enzyme is mos s able unde
somewha acidic condi ions, while i is mos ac i e a ela i ely
high pH. S eady s a e kine ic analyses e ealed he highes
ac i i y and speci ici y owa d N-ace yla ed monosaccha ides
(N-ace yl-D-glucosamine and N-ace yl-D-galac osamine) and
he disaccha ide N-diace ylchi obiose. Only a mino ac i i y
was egis e ed o non-N-ace yla ed monosaccha ides (D-
glucose, D-galac ose, and D-mannose) and disaccha ides
(mal ose and cellobiose).
To iden i y he s uc u al ea u es ha dic a e he subs a e
p o ile and o une he subs a e scope by s uc u e-based
enzyme enginee ing, he c ys al s uc u e o NagOx was
de e mined. Inspec ion o he ac i e si e con i med ha he
FAD is bico alen ly bound ia 8α-N1-his idyl and 6-S-cys einyl
linkages wi h esidues His64 and Cys123. The c ys al s uc u e
also e ealed a clea sol en -exposed binding pocke in on o
he isoalloxazine moie y o he la in co ac o ha allows
binding o mono- and oligosaccha ides. We also iden i ied a
seconda y binding pocke simila o ha p esen in Chi O,
38
wi h esidue Leu251 playing a key ole in accommoda ing he
N-ace yl moie y. On he basis o hese insigh s, we designed
and p epa ed a speci ic enzyme a ian (L251R) ha displays a
mo e elaxed subs a e p e e ence. The L251R a ian had an
imp o ed ca aly ic e iciency owa d all non-ace yla ed
monosaccha ides and disaccha ides. Docking and modeling
glucose binding con i med ha he in oduced a ginine
p omo es p oduc i e binding o glucose (Figu e S5b).
Fu he mo e, he ac i i y owa d he isaccha ide mal o iose
was in oduced. Due o i s ad an age o being unc ionally
exp essed in a good yield in a bac e ial hos , NagOx is a
p omising al e na i e o enginee ing ca bohyd a e oxidases o
selec i e oxida ion o monosaccha ides and disaccha ides.
■ASSOCIATED CONTENT
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acs.biochem.2c00307.
SDS−PAGE gel pic u es (Figu e S1), edox de e mi-
na ion da a (Figu e S2), plo s wi h s eady s a e kine ic
da a (Figu e S3), size exclusion ch oma og aphy p o ile
(Figu e S4), dime ic s uc u e o NagOx (Figu e S5),
and sc eening o subs a es (Table S1) (PDF)
Accession Codes
NagOx, A3RXB7.
■AUTHOR INFORMATION
Co esponding Au ho
Ma co W. F aaije −Molecula Enzymology, G oningen
Biomolecula Sciences and Bio echnology Ins i u e, Uni e si y
o G oningen, 9747AG G oningen, The Ne he lands;
o cid.o g/0000-0001-6346-5014; Email: m.w. aaije@
ug.nl
Au ho s
Alessand o Bo e io −Molecula Enzymology, G oningen
Biomolecula Sciences and Bio echnology Ins i u e, Uni e si y
o G oningen, 9747AG G oningen, The Ne he lands;
Depa men o Biology and Bio echnology, Uni e si y o
Pa ia, 27100 Pa ia, I aly
Wahyu S. Widodo −Molecula Enzymology, G oningen
Biomolecula Sciences and Bio echnology Ins i u e, Uni e si y
o G oningen, 9747AG G oningen, The Ne he lands
La s L. San ema −Molecula Enzymology, G oningen
Biomolecula Sciences and Bio echnology Ins i u e, Uni e si y
o G oningen, 9747AG G oningen, The Ne he lands
Hen ië e J. Rozeboom −Molecula Enzymology, G oningen
Biomolecula Sciences and Bio echnology Ins i u e, Uni e si y
o G oningen, 9747AG G oningen, The Ne he lands
Rui e Xiang −Elec onic and A omic P o ein Modelling
G oup, Ba celona Supe compu ing Cen e , E-08034
Ba celona, Spain
Víc o Gualla −Elec onic and A omic P o ein Modelling
G oup, Ba celona Supe compu ing Cen e , E-08034
Ba celona, Spain; o cid.o g/0000-0002-4580-1114
And ea Ma e i −Depa men o Biology and Bio echnology,
Uni e si y o Pa ia, 27100 Pa ia, I aly; o cid.o g/0000-
0002-9523-7128
Comple e con ac in o ma ion is a ailable a :
Biochemis y pubs.acs.o g/biochemis y A icle
h ps://doi.o g/10.1021/acs.biochem.2c00307
Biochemis y 2023, 62, 429−436
434
h ps://pubs.acs.o g/10.1021/acs.biochem.2c00307
Funding
W.S.W. was sponso ed by a LPDP schola ship om he
Minis y o Finance, Republic o Indonesia. A.M. and M.W.F.
ecei ed unding om Fondazione Ca iplo (g an 2020-0894).
L.L.S., M.W.F., V.G., and R.X. ecei ed unding om he
Eu opean Union’s Ho izon 2020 esea ch and inno a ion
p og am unde G an Ag eemen 101000607 (P ojec
OXIPRO).
No es
The au ho s decla e no compe ing inancial in e es .
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