Ci a ion: Fe nandez-Lopez, L.; Roda,
S.; Gonzalez-Al onso, J.L.; Plou, F.J.;
Gualla , V.; Fe e , M. Design and
Cha ac e iza ion o In-One
P o ease-Es e ase Plu iZyme. In . J.
Mol. Sci. 2022,23, 13337. h ps://
doi.o g/10.3390/ijms232113337
Academic Edi o : Gian anco Gila di
Recei ed: 5 Oc obe 2022
Accep ed: 29 Oc obe 2022
Published: 1 No embe 2022
Publishe ’s No e: MDPI s ays neu al
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Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
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A ibu ion (CC BY) license (h ps://
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4.0/).
In e na ional Jou nal o
Molecula Sciences
A icle
Design and Cha ac e iza ion o In-One
P o ease-Es e ase Plu iZyme
Lau a Fe nandez-Lopez 1,†, Se gi Roda 2,† , Jose L. Gonzalez-Al onso 1, F ancisco J. Plou 1,
Víc o Gualla 2,3,* and Manuel Fe e 1,*
1Depa men o Applied Bioca alysis, ICP, CSIC, 28049 Mad id, Spain
2Depa men o Li e Sciences, Ba celona Supe compu ing Cen e (BSC), 08034 Ba celona, Spain
3Ins i u ion o Resea ch and Ad anced S udies (ICREA), 08010 Ba celona, Spain
*Co espondence: ic o [email p o ec ed] (V.G.); [email p o ec ed] (M.F.)
† These au ho s con ibu ed equally o his wo k.
Abs ac :
P o eases a e abundan in p oka yo ic genomes (~10 pe genome), bu hei eco e y
encoun e s exp ession p oblems, as only 1% can be p oduced a high le els; his alue di e s om
ha o simila ly abundan es e ases (1–15 pe genome), 50% o which can be exp essed a good le els.
He e, we design a ca aly ically e icien a i icial p o ease ha can be easily p oduced. The Plu iZyme
EH
1AB1
wi h wo ac i e si es suppo ing he es e ase ac i i y was employed. A Leu24Cys mu a ion
in EH
1AB1
, emodelled one o he es e ase si es in o a p o eoly ic one h ough he inco po a ion o a
ca aly ic dyad (Cys24 and His214). The esul ing a i icial enzyme, EH
1AB1C
, e icien ly hyd olysed
(azo)casein a pH 6.5–8.0 and 60–70
◦
C. The p esence o bo h es e ase and p o ease ac i i ies in he
same sca old allowed he one-po cascade syn hesis (55.0
±
0.6% con e sion, 24 h) o L-his idine
me hyl es e om he dipep ide L-ca nosine in he p esence o me hanol. This s udy demons a es
ha ac i e si es suppo ing p o eoly ic ac i i y can be a i icially in oduced in o an es e ase sca old
o design easy- o-p oduce in-one p o ease-es e ase Plu iZymes o cascade eac ions, namely, he
syn hesis o amino acid es e s om dipep ides. I is also possible o design a i icial p o eases wi h
good p oduc ion yields, in con as o na u al p o eases ha a e di icul o exp ess.
Keywo ds:
es e ase; Plu iZyme; p o ease; p o ein enginee ing; compu a ional chemis y; cascade e-
ac ion
1. In oduc ion
One-po cascade eac ions a e chemical p ocesses highly appealing o he indus ial
sec o , as hey allow he syn hesis o complex p oduc s, s a ing om ela i ely simple eac-
ion condi ions [
1
–
4
]. Ne e heless, he implemen a ion o hese eac ions is a demanding
ask, and usually, i equi es he enginee ing o each ca alys om each in e nal chemical
eac ion. Mo eo e , ca alys s mus be speci ic o hei eac an , a oiding unpleasan side
p oduc s as much as possible. Fo ha eason, enzymes a e excellen candida es o se up
mul is ep andem eac ions, since hey wo k unde milde condi ions han ino ganic ca a-
lys s and a e also egio- and s e eo-selec i e/speci ic [
2
–
4
]. Addi ionally, ecen ad ances in
he a ional design and di ec ed e olu ion o enzymes ha e in oduced a signi ican success
a e in imp o ing di e se enzyma ic p ope ies, allowing hem o compe e wi h con en-
ional ca alys s [
2
–
5
]. Rema kably, cu en de elopmen s include designing enzymes wi h
new- o-na u e ca aly ic ac i i ies, such as ca bene ans e in an enginee ed cy och ome
P450 enzyme [
6
], expanding he a ie y o he cascade eac ions we can ackle. Ano he
in e es ing example o he usage o enzymes o cascade eac ions is he one de eloped by
Me ck & Co., o syn hesize isla a i (a po en ial d ug o HIV ea men ) om ela i ely
simple building blocks [7].
Pe o ming he cascade eac ion wi hin a single enzyme in oduces complexi ies. Fo
his eason, he s udy and design o bioca alys s pe o ming di e en chemical eac ions
In . J. Mol. Sci. 2022,23, 13337. h ps://doi.o g/10.3390/ijms232113337 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 13337 2 o 14
in ei he he same p o ein sca old [
8
–
13
] o by linking mul iple domains [
14
–
17
] ha e
become a ho opic in p o ein enginee ing h ough a numbe o s a egies [
18
,
19
]. One
o hese s a egies is o bene i om he capabili ies o p o ein enginee ing suppo ed by
he compu a ional esou ces o he design o a i icial enzymes wi h a supe io o no el
pe o mance, compa ed o na u al enzymes. Beyond he enginee ing o enzymes by hese
echniques, he possibili y o inco po a ing ac i e cen e s in o p o ein sca olds opens he
oppo uni y o design a i icial bioca alys s. A numbe o compu a ional me hods ha e been
success ully applied o in oduce biological ac i e si es in o p o ein sca olds. They include,
he Rose a-like me hods and he P o ein Ene gy Landscape Explo a ion (PELE) so wa e,
h ough which biological si es suppo ing es e -hyd olysis ha e been inco po a ed in
di e en p o ein sca olds [
8
,
9
,
19
]. Howe e , PELE is he only desc ibed me hod o gene a e
se e al a i icial biological ac i es si es in he same enzyme sca old, which open a ange o
possibili ies in cascade eac ions, as demons a ed using di e en enzyme sca olds in o
which wo same o di e en biological ac i i ies we e in oduced [
8
,
20
]. These wo-ac i e
si es enzymes, named Plu iZymes, could in oduce an ideal scena io o one-po cascade
eac ions, by educing he cos s o p oducing wo enzymes sepa a ely. In some cases, hei
use may also allow inc eased yields by acili a ing he ans e o eac ion in e media es
be ween ac i e si es wi hin he same p o ein, compa ed o he ans e be ween si es o
di e en p o eins [
20
]; howe e , his will depend on he a chi ec u e and posi ioning o
hese si es.
One example is ou ecen wo k building Plu iZymes in wo di e en enzyme amilies.
Fi s , we de eloped an es e ase, EH
1AB1
, whe e a second ca aly ic iad (Se -His-Asp/Glu)
was added, c ea ing an enzyme wi h a i icial and na i e ac i e si es suppo ing es e hy-
d olysis (T
op
o 8–45
◦
C) [
8
]. Impo an ly, he na i e ac i e si e could be ans o med in o a
me al-complex chemoca aly ic si e by adding a suicide inhibi o , allowing he oxida ion and
F iedel–C a s alkyla ion eac ions. Thus, one-po cascade eac ions could be cons uc ed
om his bioca alys [
8
]. Second, we ook an
ω
- ansaminase and added an a i icial si e
suppo ing es e hyd olysis h ough he in oduc ion o a ca aly ic iad (Se -His-Asp/Glu).
A polypep ide ha ing wo bio ic si es ca alysing di e en ypes o chemical eac ions was
hus designed, which could ans o m oxo-es e s in o amino acids in a one-po eac ion [
20
].
In he p esen s udy, we aimed a a di e en app oach, pushing he limi s o a hyd olase
si e by adding ex a biochemis y h ough he addi ion o a cys eine-his idine ca aly ic
dyad. Using ou Plu iZyme EH
1AB1
, we in oduced he p o ease ac i i y by designing
a single mu an , Leu24Cys, which was capable o ecycling a his idine esidue, His214,
om an al eady exis ing ca aly ic iad. A p io i, compu a ional analyses indica e ha he
mu a ion should no dis up he es e ase ac i i y o he ecycled es e ase si e. The e o e,
he newly designed Plu iZyme, he ein e e ed o as EH
1AB1C
, included h ee po en ial si es.
The i s suppo s es e hyd olysis h ough a na i e ca aly ic iad (Se 161, Asp256 and
His286) and an oxyanion hole (Gly88, Gly89 and Gly90), wi h Se 161 being he nucleophile.
The second, also suppo ing es e hyd olysis, would employ an a i icial ca aly ic iad
(Se 211, Asp25 and His214) wi h Se 211 as he nucleophile and an oxyanion hole (Gly207,
Ty 208 and Phe209). The hi d would suppo he p o ease ac i i y h ough a ca aly ic dyad
(Cys24 and His214).
Adding a si e suppo ing p o eoly ic ac i i y was a ge ed, as p o eases a e pi o al
enzymes o he hyd olysis o pep ide bonds in ma e ials whe e p o eins a e abundan
componen s and a e also widely used in o ganic syn hesis [
21
]. This is why hey cons i u e
60–65% o he global indus ial ma ke , g owing a an annual g ow h a e o 5.6% [
22
].
Th ough e olu ion, p o eases ha e adap ed o he wide ange o condi ions ound in
complex o ganisms ( a ia ions in pH, educ i e en i onmen , e c.) and use di e en
ca aly ic mechanisms o subs a e hyd olysis [
23
]; hei mechanism o ac ion classi ies hem
as ei he se ine, cys eine o h eonine p o eases (amino- e minal nucleophile hyd olases) o
aspa ic, me allo and glu amic p o eases (wi h glu amic p o eases being he only sub ype
no ound in mammals hus a ) [
24
]. P o eases speci ically clea e p o ein subs a es ei he
om he N o C e mini (aminopep idases and ca boxypep idases, espec i ely) and/o in
In . J. Mol. Sci. 2022,23, 13337 3 o 14
he middle o he molecule (endopep idases). P o eases can be easily sc eened by unc ional
sc eens o in silico p edic ions in mic oo ganisms o mic obial communi ies by applying
genomic and me agenomic app oaches [
22
]. O cou se, i should be s essed ha he
no el y i sel does no gua an ee a be e enzyma ic pe o mance and be e oppo uni ies
o comme cializa ion, whose analysis equi es labo ious we lab wo k. This is no a i ial
exe cise, gi en ha no all genes in a genome o a me agenome can be success ully cloned
and exp essed. This is especially impo an in he cases o p o eases ha su e majo
p oblems o exp ession, compa ed o o he ypes o enzymes ha a e as easy o be sc eened
as p o eases bu be e o be p oduced a high le els [
22
]. P o eases a e used in a b oad
ange o applica ions, including bio e ine ies a ge ing a b oad ange o biomasses [
22
].
Howe e , he known and new p o eases posi i ely impac addi ional p ocesses, such as
cascade eac ions whe e p o eases ha e a pi o al ole [
25
]. He e, we a ge ed a cascade
eac ion in ol ing pep ide bond clea age and es e bond o ma ion and demons a ed
ha he newly designed EH
1AB1C
Plu iZyme was capable o con e ing he dipep ide L-
ca nosine (
β
-alanine-L-his idine) in o L-his idine me hyl es e , an in e media e o he
design o Schi base ligands [26].
2. Resul s
2.1. Molecula Simula ions
This wo k is based on he design o Plu iZymes (la in oo plu i: mul iplici y), an
enzyme design in which a single polypep ide ha bou s wo di e en ac i e cen e s, one
na i e and one a i icial [
8
,
20
]. The idea is based on loca ing a na i e enzyme, h ough he
P o ein Ene gy Landscape Explo a ion (PELE) so wa e, exis ing binding pocke s whe e
a a ge subs a e could be accommoda ed and u ning hem in o ca aly ic ac i e si es by
in oducing all he esidues needed o he ca alysis. Following om his, we ha e ecen ly
success ully ound and designed, by in oducing a ew mu a ions, a second a i icial ac i e
si e (Se -His-Asp) in an es e ase con aining a na i e si e o gene a e a Plu iZyme, EH
1AB1
,
wi h wo e icien biological ac i e si es o he es e hyd olysis ha coexis s in a close
egion. Compa ed o he na u al iad ( e e ed o as EH
1A1
), he newly in oduced a i icial
si e ( e e ed o as EH
1B1
) is sligh ly mo e sol en exposed and loca ed a an ~10 A dis ance
(Figu e 1A). We ha e conside ed inc easing he numbe o chemical eac ions wi hin his
enzyma ic design. The compu a ional p o ocol employed he ein is simila o he one used
in ou Plu iZyme designs, la gely desc ibed in [
8
,
9
,
19
,
27
], bu we ha e ocused mo e on a
local egion explo a ion.
Figu e 1.
(
A
) Slice o he 3D s uc u e o EH
1AB1
ep esen ing he main and a i icial ac i e si es.
The C a oms a e s ained in ma oon and da k g een in he main ac i e si e and a i icial ac i e si e,
espec i ely. (
B
) Rep esen a i e binding pose o glyce yl ip opiona e (wi h C a oms s ained in g ay)
whe e we can see he posi ion o Leu24. Leu24 (in yellow) o ms a hyd ogen bond wi h he ca bonyl
O a om in he es e bond by he NH g oup in he backbone (indica ed by a cyan dashed line) and is
close o he His214 esidue.
We i s a emp ed o pe o m an addi ional global su ace explo a ion o loca ing
po en ial si es suppo ing he es e hyd olysis using glyce yl ip opiona e as a p obe. As
shown in Figu es S1 and S2, we did no ob ain addi ional al e na i es. We hen conside ed
he inco po a ion o a new ac i e cen e o suppo ano he eac ion, namely, p o eolysis,
In . J. Mol. Sci. 2022,23, 13337 4 o 14
o explo e he possibili y o designing an in-one p o ease-es e ase Plu iZyme. Fo his,
we ocused on he local analysis o he ca aly ic iad egions (Figu e 1A), whe e we
clea ly obse ed how he mo e exposed si e, EH
1B1
, could accommoda e bulkie subs a es
(as in he case o pep ides). Following he inspec ion o he bes enzyme-subs a e poses
(Figu e 1B), we decided o in oduce he Leu24Cys a ian , named EH
1AB1C
, which, oge he
wi h he ca aly ic his idine o EH
1B1
, His214, could in oduce a ca aly ic dyad simila o
hose seen in p o eases.
We p oceeded by p epa ing mul iple dipep ides, namely, AH, AQ, DI, EA, FF, KA,
LA, LL, NV, PF, QQ, RG, SW, TM, YN and YY, and pe o med a local PELE explo a ion
o each dipep ide. By doing so, we can model he p opensi y o o m ca aly ically ac i e
posi ions be ween each pep ide bond and he newly designed ca aly ic dyad: Cys24 and
His214. He e, we we e no sea ching o he bes pep ide bu o see i a di e se se o hem
could each ca aly ically ac i e con o ma ions. In e es ingly, as shown in Figu e 2, Table S1,
and Figu es S3–S5, all dipep ides, excep o YY we e able o ind he EH
1B1
si e and each
ca aly ic poses a ound he enginee ed cys eine-his idine dyad. Likewise, i can be seen ha
he ca aly ic hyd ogen bond dis ances (Cys o His) we e qui e good du ing he simula ion
(Table S2, Figu es S6 and S7).
Figu e 2.
PELE’s ene gy analysis and ep esen a i e geome ies. EH
1AB1C
densi y plo s o he
dis ibu ion o he ca aly ic cys eine-subs a e dis ances agains he in e ac ion ene gy o he bes -
bound dipep ide subs a e (PF, op) and he wo s one (YY, bo om). Only he 10% lowes pe cen ile,
ega ding he dis ance is shown. In he igh panel, we display ep esen a i e ca aly ic poses o he
dipep ide subs a e in he pu a i e p o ease si e. The C a oms a e s ained in ma oon, da k g een and
yellow in he EH
1A1
ac i e si e esidues, EH
1B1
and Cys24, espec i ely, and each subs a e is shown
using he densi y plo colou (in whea o PF and blue o YY). The ene gy p o iles we e c ea ed wi h
he Ma plo lib lib a y [28].
2.2. Expe imen al Valida ion: EH1AB1C Is an E icien P o ease
The ecombinan mu an , he ea e e e ed o as EH
1AB1C
, was success ully exp essed
in soluble o m in Esche ichia coli and pu i ied by nickel a ini y ch oma og aphy. The
pu i ied p o ein (app ox. 10 mg pe li e o cul u e) was desal ed by ul a il a ion, and i s
p o eoly ic ac i i y was es ed h ough a gene al luo escence assay, namely, he BODIPY-
FL-casein assay using he EnzChek
®
P o ease Assay Ki , which is insensi i e o pH changes.
In . J. Mol. Sci. 2022,23, 13337 5 o 14
Fi s , he pH p o ile o he enzyme was ob ained (Figu e 3A). I s op imal pH o ac i i y
was 7.0, e aining mo e han 70% o he maximal ac i i y a pH alues om 6.5 o 8.0. We
hen analysed i s empe a u e p o ile using he ch omogenic subs a e azocasein. A pH 7.0,
EH
1AB1C
showed maximal ac i i y a 70–75
◦
C, e aining mo e han 70% o he maximum
ac i i y a 50–85
◦
C (Figu e 3B). The speci ic ac i i y o EH
1AB1C
was compa ed o ha o
he comme cial p o ease Neu ase 0.8 L (No ozymes A/S, Bags ae d, Denma k). A pH
7.0 and 30
◦
C, he speci ic ac i i y o EH
1AB1C
was 2.63
±
0.06 U/mg p o ein, while ha
o he comme cial Neu ase 0.8 L was 1.86
±
0.11 U/mg p o ein. No e ha he o iginal
design, EH
1AB1
, did no show any p o eoly ic ac i i y wi h BODIPY-FL-casein o azocasein,
demons a ing ha he inco po a ion o he dyad Cys24-His214 in oduced p o eoly ic
ac i i y.
Figu e 3.
pH and empe a u e p o iles o he pu i ied EH
1AB1C
. (
A
) pH p o ile a 30
◦
C. (
B
) Tempe a-
u e p o ile a pH 7.0. The maximal ac i i y was de ined as 100%, and he ela i e ac i i y is shown as
he pe cen age o he maximal ac i i y (mean
±
SD o iplica es), de e mined unde s anda d eac ion
condi ions wi h BODIPY-FL-casein (in panel (
A
)) and azocasein (in panel (
B
)) as he subs a es. The
g aphics we e c ea ed wi h Excel e sion 14.0.
The es e ase ac i i y o he EH
1AB1C
mu an was quan i ied a 30
◦
C and pH 8.0 wi h
he model es e glyce yl ip opiona e and compa ed o ha o he ini ial cons uc EH
1AB1
.
This subs a e can be con e ed by bo h es e ase si es ( he na i e and he a i icial si es).
We ound ha he EH
1AB1C
mu an is an e icien es e ase capable o hyd olysing his es e
a 3160
±
76 U/g. Howe e , we obse ed ha his alue was only 14.5% o ha o EH
1AB1
.
To e alua e whe he his educ ion could be because he a i icial es e ase cen e (B si e),
du ing i s emodelling o add a p o ease cen e , has been al e ed and wi h i , i s ac i i y,
would equi e e alua ing he ac i i y wi h B-si e speci ic es e s. Howe e , all es ed es e s
hyd olysed by his si e a e also hyd olysed by he na i e es e ase cen e (A-si e), due
o he b oad subs a e speci ici y o he la e [
8
]. The e o e, we canno be su e whe he
he obse ed educ ion in ac i i y is due o a possible e ec o he mu a ion in oduced
(Leu24Cys) on he a chi ec u e o he B-cen e , o a local e ec on he s uc u e, o o he
possibili y o pa ial au olysis (sel -diges ion) by he addi ion o he p o ease si e.
2.3. Applica ion o EH1AB1C in a One-Po Cascade Reac ion
Following he compu a ional design o a p o ease si e in EH
1AB1
and he cha ac e -
iza ion o he success ul a ian EH
1AB1C
, we wan ed o es he abili y o his alida ed
Plu iZyme o ca alyse a cascade eac ion o in e es . As a model eac ion, we chose o syn-
hesize L-his idine me hyl es e , an in e media e o he design o Schi base ligands [
26
],
om he dipep ide L-ca nosine (β-alanine-L-his idine) (Figu e 4).
In . J. Mol. Sci. 2022,23, 13337 6 o 14
The simula ion o he eac ion by a local PELE explo a ion showed e icien L-alanine-
L-his idine (AH) ca aly ic (hyd oly ic) binding poses a he p o eoly ic si e (Table S1).
We also ob ained good ca aly ic poses o L-ca nosine (
β
-alanine-L-his idine) (Figu e 5;
Table S1). Thus, we expec ed ha L-ca nosine would be hyd olysed a he p o ease si e,
due o i s simila i y wi h he L-alanine-L-his idine. We se up wo eac ions a 40
◦
C and
pH 7.0. The i s one con ained L-ca nosine (5 mM) in he bu e , 40 mM HEPES, a pH 7.0.
The second one ep esen ed a one-po cascade eac ion wi h all he eagen s necessa y o
he hyd olysis o L-ca nosine and he es e i ica ion o he co esponding eac ion p oduc s
wi h me hanol, i.e., L-ca nosine and me hanol. Following he addi ion o he E. coli cells
exp essing he EH
1AB1C
Plu iZyme, he le els o he subs a e L-ca nosine, he in e media es
β
-alanine and L-his idine, and he possible p oduc s
β
-alanine me hyl es e and L-his idine
me hyl es e , we e quan i ied using high-pe o mance liquid ch oma og aphy (HPLC) o e
20 h (Figu e 6and Figu e S8). No e ha o hese es s, cells exp essing EH
1AB1C
we e used
ins ead o pu i ied p o ein o inc ease he s abili y o he bioca alys s, which in soluble o m
may be inac i a ed.
Figu e 4.
Schema ic ep esen a ion o he main p oduc ob ained in a one-po eac ion wi h he
dipep ide L-ca nosine (
β
-alanine-L-his idine) and me hanol. A schema ic ep esen a ion o he
wo eac ion in e media es and he wo possible p oduc s (
β
-alanine and L-his idine me hyl es e s)
is shown. As shown in he igu e, he p e e en ial ou e ound is he p oduc ion o L-his idine
me hyl es e ia he hyd olysis o L-ca nosine a he p o ease si e and he selec i e es e i ica ion
wi h me hanol o L-his idine (bu no
β
-alanine) a he es e ase si e. No app eciable o ma ion o
L-ca nosine me hyl es e and
β
-alanine me hyl es e (g ay colou ) was de ec ed. The igu e was
c ea ed using ChemD aw 18.2.
In . J. Mol. Sci. 2022,23, 13337 7 o 14
Figu e 5.
EH
1AB1C
densi y plo o he dis ibu ion o he ca aly ic cys eine-subs a e dis ance agains
he in e ac ion ene gy o L-ca nosine. Only he 10% lowes pe cen ile, ega ding he dis ance is
shown. In he igh panel, we display he ep esen a i e ca aly ic poses o L-ca nosine in he pu a i e
p o ease si e. The C a oms a e s ained in ma oon, da k g een, and yellow in he EH
1A1
ac i e si e
esidues, EH
1B1
, and Cys24, espec i ely, and L-ca nosine is shown using he pink colou used in he
densi y plo . The ene gy p o iles we e c ea ed wi h he Ma plo lib lib a y [28].
We ound ha he L-ca nosine dipep ide (concen a ion o 5 mM) was ully con e ed
(>93.0 ±0.1%) a e 20 h in he bu e , ob aining β-alanine (4.5 ±0.1 mM) and L-his idine
(4.75
±
0.05 mM) (Figu e 6). When he eac ion was pe o med in me hanol, L-ca nosine
was con e ed a e 20 h (90.5
±
12.3%), wi h he main p oduc s being
β
-alanine (4.5
±
0.1 mM), L-his idine (1.8
±
0.1 mM) and L-his idine me hyl es e (2.8
±
0.5 mM).
β
-
Alanine me hyl es e and L-ca nosine me hyl es e we e no ound as p oduc s. This esul
demons a es ha L-ca nosine is hyd olysed a he a i icial p o eoly ic si e; u he mo e,
he es e ase si e only ecognized L-his idine bu no
β
-alanine as a subs a e, which is
es e i ied o yield L-his idine me hyl es e as he only amino acid es e .
Figu e 6.
Concen a ions o he subs a es, in e media es and inal p oduc s ob ained o he con-
e sion o L-ca nosine by he EH
1AB1C
Plu iZyme in he absence (
A
) o p esence (
B
) o me hanol.
Reac ion condi ions in A: [cells exp essing EH
1AB1C
]: 5
µ
L o esuspended cells a OD
600 nm
o
15.0; [L-ca nosine]: 5 mM; bu e : 92.5
µ
L 40 mM HEPES, pH 7.0; eac ion inal olume: 100
µ
L.
Reac ion condi ions in A: [cells exp essing EH
1AB1C
]: 5
µ
L o esuspended cells a OD
600 nm
o 15.0;
[L-ca nosine]: 5 mM; sol en : 92.5
µ
L me hanol; eac ion inal olume: 100
µ
L (92.5% me hanol).
No e, ha he amoun o he
β
-Ala me hyl es e was below he de ec ion limi unde ou expe imen al
condi ions, and his is why yellow ba s a e no isible in he igu e. The igu e was c ea ed using
SigmaPlo 14.0 so wa e.
To highligh , we ound ha when using as bioca alys cells exp essing he o iginal
EH
1AB1
Plu iZyme, L-ca nosine was no con e ed o any o he in e media es o inal
p oduc s (Figu e S9A); his indica es ha he p o eases in E. coli do no a ec he esul s
and ha in he absence o he a i icial p o eoly ic cen e , he cascade eac ion is no easible.
Fu he , bo h he o iginal EH
1AB1
Plu iZyme and he mu an EH
1AB1C
Plu iZyme bioca alys s
In . J. Mol. Sci. 2022,23, 13337 8 o 14
we e shown o con e L-his idine o L-his idine me hyl es e in he p esence o me hanol,
bu no
β
-alanine o he co esponding me hyl es e (Figu e S9B,C). These con ol es s,
oge he wi h he esul s p esen ed in Figu e 6, demons a ed ha he inco po a ion o
he dyad Cys24-His214 in he EH
1AB1C
Plu iZyme was esponsible o he p o eolysis o
L-ca nosine, and ha he es e ase(s) ac i e si e(s) o iginally p esen in EH
1AB1C
suppo ed
he acyla ion o L-his idine and he p oduc ion o he amino acid es e (L-his idine me hyl
es e ).
3. Discussion
P o eases ha can b eak es e bonds and es e ases and lipases ha can b eak amide
bonds exis in na u e; hey a e an example o ca aly ically p omiscuous enzymes [
29
–
34
].
The p esence o a Se o a Cys ca aly ic esidue, along wi h di e ences in he subs a e
binding si e, seems o be he main eason o subs a e disc imina ion and he p omis-
cuous beha io . When a emp ing o enginee such ac i i y om sc a ch (ab ini io) o
when in oducing enough mu a ions o accommoda e a dyad/ iad, we ypically ind low
ac i i y p o iles [
35
–
37
]. Ne e heless, hese esul s ha e pa ed he way o mo e ecen
implemen a ions, such as ou ecen Plu iZymes, whe e he ex ensi e in silico op imiza ion
(ca aly ic dis ances) has p o ided wide subs a e p omiscui y, high subs a e con e sion
a es, and e en he de elopmen o cascade eac ions [
8
,
9
]. In his s udy, we in oduced
he p o ease ac i i y by eusing a (p e iously enginee ed) es e ase si e. We ecycled he
ca aly ic his idine and inse ed only a cys eine esidue, achie ing high ca aly ic a es in
he s anda d pep ide assays. As in ou p e ious designs, he po en ial o unning accu a e
enzyme-subs a e induced i simula ions p o ided enough insigh s. Thus, hese esul s
again illus a e how simula ion echniques using molecula modelling a e ma u e enough
and capable o p o iding a ealis ic desc ip ion o localized changes. Such modelling po-
en ial, along wi h he p opensi y o ca aly ic dyads/ iads o o m (s abilizing) hyd ogen
bonds, makes he design o hyd olase ac i e si es an a o dable ask; we ha e succeeded in
all ecen a emp s [8,9,20].
The main eason behind ou e o s de i es om ou goals o de eloping single sca -
old enzymes wi h mul iple biochemical ac i i ies ha a e capable o pe o ming one-po
cascade eac ions. P e ious a emp s in he na i e es e ase EH
1
ocused on in oducing a
second a i icial es e ase ac i e si e and he inco po a ion o an i e e sible-linked inhibi o
con aining a me a-chela ing moie y o one o he si es, allowing he addi ion o a i icial
abio ic oxida i e chemis y as a complemen o he o iginal bio ic es e ase ac i i y [
8
]. Mo e
ecen ly, we we e able o add a i icial bio ic es e ase ac i i y in o an
ω
- ansaminase [
20
].
Wi h ou new ca aly ically e icien design, EH
1AB1C
, we a e now capable o b eaking
amide bonds and o ming es e bonds o yield na u al and nonna u al amino acid es-
e s, he ein exempli ied by L-his idine me hyl es e , which a e impo an in e media es
in o ganic syn hesis [
38
]. Amino acid me hyl es e s, such as L-his idine me hyl es e , can
be chemically syn hesized a oom empe a u e h ough he es e i ica ion o L-his idine
wi h ime hylchlo osilane (TMSCl) and me hanol a high yields (88–96%, using 10 mM
L-his idine) [
38
]; he bioca alys s epo ed he ein o e milde and en i onmen ally iendly
condi ions and good yields o he syn hesis o amino acid es e s use ul in he pha maceu i-
cal indus y. In addi ion, bo h p o ease and es e ase si es in EH
1AB1C
may o e speci ici y,
as shown he e, o example, by he capaci y o he la e o es e i y L-his idine bu no
β
-alanine. The capaci y o some p o eases o ca alyse es e hyd olysis has been used o
p oduce amino acid es e s wi h a high op ical pu i y h ough he selec i e hyd olysis o
D,L-amino acid es e s (e.g., me hyl and benzyl es e s) o p o ide, o example, L-amino
acids and op ically pu e D-amino acid es e s [
39
,
40
]. Howe e , in his case, an amino acid
es e is needed as he ini ial subs a e, in con as o ou Plu iZyme, which can di ec ly use
dipep ides and possibly longe oligopep ides.
We would like o highligh ha he eac ion condi ions o he subs a e epo ed in
his wo k and o he s ye o be es ed ha e no been op imized. Thus, his design o e s
in e es ing p ope ies o be u ilized in indus ial se ings, such as an in eg a ed bio e ine y
In . J. Mol. Sci. 2022,23, 13337 9 o 14
o biomass eco e y based on p o eases o he p oduc ion o p o ein hyd olysa es, bioac-
i e pep ides, and amino acids applicable o a wide ange o applica ions. I also o e s a
pla o m o syn hesize a wide ange o biobased p oduc s h ough in-one cascade eac ions
in ol ing es e and amine bond hyd olysis and o ma ion.
4. Ma e ials and Me hods
4.1. Ma e ials
Azocasein ( e . A2765-1G),
β
-alanine ( e . 146064-25G), L-his idine ( e . H7750-25G),
L-his idine me hyl es e ( e . H15403-25G),
β
-alanine me hyl es e ( e . 05210-10G), L-
ca nosine ( e . C9695-10MG), glyce yl ip opiona e ( e . W328618-1KG-K), and me hanol
( e . 34966-1L) we e o de ed om Me ck Li e Science S.L.U. (Mad id, Spain). The EnzChek
®
P o ease Assay Ki ( e . E6638) was p o ided by In i ogen, The mo Fishe Scien i ic Inc.,
Wal ham, MA, USA. FMOC chlo ide ( e . GE3236-1G) was pu chased om Glen ham Li e
Sciences, Co sham, UK.
4.2. P o ein and Chemical P epa a ion o he In Silico Analysis
The apo EH
1A
c ys al s uc u e (5JD4) and he holo EH
1AB1
c ys al s uc u e (6RB0)
we e p epa ed and p o ona ed a pH 8.0, he pH a which he expe imen al assays we e
pe o med, using P o ein P epa a ion Wiza d [
41
]. This includes ixing side chains and
loops missing in he c ys al s uc u e using P ime so wa e [
42
]. The es e compound
used as a p obe o ind nonca aly ic hyd olase si es was glyce yl ip opiona e; he pep ide
binding assays o he gene a ed a ian used 16 dipep ides (AH, AQ, DI, EA, FF, KA, LA,
LL, NV, PF, QQ, RG, SW, TM, YN, and YY) as subs a es. All subs a es we e modelled using
he OPLS2005 o ce ield [
43
]. The a omic cha ges o glyce yl ip opiona e and he ca aly ic
se ine esidues bound wi h he me hyl hyd ogen (R)-hexylphosphona e inhibi o [
8
,
20
]
we e calcula ed wi h Jagua [
44
] using densi y unc ional heo y wi h a B3LYP-D3 exchange-
co ela ion unc ional and he pola ized iple-ze a (pVTZ) basis se .
4.3. P o ein Ene gy Landscape Explo a ion (PELE) Simula ions
PELE was used o ind he nonca aly ic pep ide binding si es in EH
1A
/EH
1AB1
and
check i he ca aly ic poses can be eached in he unc ionalized a ian [
8
,
9
,
19
,
27
]. PELE is a
Mon e Ca lo (MC)-based algo i hm coupled wi h p o ein s uc u e p edic ion me hods [
45
].
The heu is ic MC app oach begins wi h he sampling o di e en mic os a es by ini ially
applying small pe u ba ions ( ansla ions and o a ions) on he ligand. Then, he lexibili y
o he p o ein is aken in o accoun by applying no mal modes h ough he aniso opic
ne wo k model (ANM) app oach. Once he sys em has been pe u bed, side chains o he
esidues nea he ligand a e sampled wi h a lib a y o o ame s o a oid s e ic clashes.
Finally, a unca ed New on minimiza ion wi h he OPLS2005 o ce ield [
43
] is pe o med,
and he new mic os a e is accep ed o ejec ed, acco ding o he Me opolis c i e ion. The
a iable dielec ic gene alized Bo n non-pola (VDGBNP) implici sol en [
46
] was applied
o mimic he in luence o wa e a ound he p o ein.
4.4. P edic ion o ∆∆G in he EH1AB1 Va ian
The
∆∆
G(mu -WT) o s abili y in he expe imen ally es ed a ian s was calcula ed
using he module o he modynamic s abili y om Ho Spo Wiza d, which uses FoldX
o epai possible p oblems in he p o ein s uc u e and Rose a o pe o m he ene gy
minimiza ion and ∆∆G calcula ion (acco ding o p o ocol 3 om Rose a) [47].
4.5. Sou ce and P oduc ion o EH1AB1C
The sequence o EH
1AB1C
was syn hesized by GenSc ip Bio ech (GenSc ip Bio ech,
EG Rijswijk, Ne he lands) and was codon-op imized o maximize he exp ession in E. coli.
The gene was lanked by BamHI and HindIII (s op codon) es ic ion si es and inse ed
in o a pET-45b(+) exp ession ec o wi h an ampicillin selec ion ma ke (GenSc ip Bio ech,
EG Rijswijk, Ne he lands), which was u he in oduced in o E. coli BL21(DE3). The
