Regis e ed cha i y numbe : 207890
As ea u ed in:
See Elisabe Pi es, Vicen Moline ,
Fe nando López-Gallego e al.,
Chem. Sci., 2020, 11, 12009.
Showcasing esea ch om P o esso López-Gallego’s
labo a o y, He e ogeneous Bioca alysis G oup, Cen e o
Coope a i e Resea ch in Bioma e ials (CIC biomaGUNE)
Basque Resea ch and Technology Alliance (BRTA),
Donos ia-San Sebas ián, Spain.
Selec i e oxida ion o alkyl and a yl glyce yl monoe he s
ca alysed by an enginee ed and immobilised glyce ol
dehyd ogenase
A new a ian o glyce ol dehyd ogenase om Bacillus
s ea o he mophilus has been enginee ed and immobilised on
po ous mic obeads o accep a ba e y o alkyl/a yl glyce yl
monoe he s as subs a es. QM/MM compu a ional s udies
e eal he excellen S-enano oselec i i y owa ds he glyce ol
de i a i es and si e-di ec ed immobilisa ion echniques allow
he pe o ming o kine ic esolu ion o acemic (R)-3-(alkoxy/
a yloxy)p opan-1,2-diols- This he e ogeneous bioca alys is
e-usable and can be coupled wi h an NAD+ ecycling sys em
o in ensi y he p ocess.
sc.li/chemical-science
Selec i e oxida ion o alkyl and a yl glyce yl
monoe he s ca alysed by an enginee ed and
immobilised glyce ol dehyd ogenase†‡
Susana Velasco-Lozano,
a
Mai e Roca,
b
Alejand o Leal-Duaso,
a
Jos´
e A. Mayo al,
ac
Elisabe Pi es, *
ac
Vicen Moline *
b
and Fe nando L´
opez-
Gallego *
ade
Enzymes ac ing o e glyce yl e he s a e sca ce in li ing cells, and consequen ly bioca aly ic ans o ma ions
o hese molecules a e a e despi e hei in e es o indus ial chemis y. In his wo k, we ha e enginee ed
and immobilised a glyce ol dehyd ogenase om Bacillus s ea o he mophilus (BsGlyDH) o accep a ba e y
o alkyl/a yl glyce yl monoe he s and ca alyse hei enan ioselec i e oxida ion o yield he co esponding 3-
alkoxy/a yloxy-1-hyd oxyace ones. QM/MM compu a ional s udies deciphe he key ole o D123 in he
oxida ion ca aly ic mechanism, and e eal ha his enzyme is highly enan ioselec i e owa ds S-isome s
(ee > 99%). Th ough s uc u e-guided si e-selec i e mu agenesis, we find ha he mu a ion L252A
sculp s he ac i e si e o accommoda e a p oduc i e configu a ion o 3-monoalkyl glyce ols. This
mu a ion enhances he k
ca
163- old owa ds 3-e hoxyp opan-1,2-diol, esul ing in a specific ac i i y
simila o he one ound o he wild- ype owa ds glyce ol. Fu he mo e, we immobilised he L252A
a ian o in ensi y he p ocess, demons a ing he eusabili y and inc easing he ope a ional s abili y o
he esul ing he e ogeneous bioca alys . Finally, we manage o in eg a e his immobilised enzyme in o
a one-po chemoenzyma ic p ocess o con e glycidol and e hanol in o 3-e hoxy-1-hyd oxyace one
and (R)-3-e hoxyp opan-1,2-diol, wi hou affec ing he oxida ion ac i i y. These esul s hus expand he
uses o enginee ed glyce ol dehyd ogenases in applied bioca alysis o he kine ic esolu ion o glyce ol
e he s and he manu ac u ing o subs i u ed hyd oxyace ones.
In oduc ion
Enzymes ac ing o e glyce yl e he subs a es a e sca ce in cell
me abolisms. Among he low di e si y o biochemical eac ions
in ol ing glyce yl e he s, we highligh lignin biosyn hesis,
1
he
me abolic syn hesis o e he lipids
2
and plasmalogens
3–5
ha
play impo an physiological oles.
6
Despi e he low abundance o glyce ol de i ed alkyl e he s in
na u e, hese molecules a e eno mously a ac i e o he
chemical indus y o manu ac u e bo h commodi ies
7
(i.e. uels
and sol en s)
8
and specialised p oduc s (i.e. cosme ics and
d ugs).
9–11
In pa icula , monoalkyla ed glyce ol e he s a e
conside ed among he mos p omising a ge s o alo ise he
su plus o glyce ol om bio ene y ac i i ies.
12–14
The chemical
s uc u e o many app o ed and comme cial d ugs inco po a es
a glyce ol skele on wi h a leas one alkoxy o a yloxy subs i uen
(Scheme 1A). Some o hese d ugs a e adminis e ed as pu e
enan iome s which encou ages medical chemis s o de elop
enan ioselec i e syn he ic schemes. He ein, bioca alysis bu s s
as a key enabling echnology in chemical manu ac u ing due o
he exquisi e egio- and enan ioselec i i y o enzymes.
In he las decade, he pha maceu ical indus y is success-
ully in eg a ing mo e bioca aly ic s eps in hei d ug syn he ic
p ocesses; new enzyme ou es o he manu ac u ing o si a-
glip in
15
and isla a i
16
illus a e his end.
Syn hesis o monoalkyl/a yl glyce ols ha e been a ained
h ough many diffe en chemical me hodologies, including
bo h s oichiome ic and ca aly ic ones.
17
Recen ly, Leal-Duaso
a
Ca ´
alisis He e og´
enea en S´
ın esis O g´
anicas Selec i as, Ins i u o de S´
ın esis Qu´
ımica y
Ca ´
alisis Homog´
enea (ISQCH-CSIC), Uni e si y o Za agoza, Ped o Ce buna, 12,
50009, Za agoza, Spain. E-mail: [email protected]; [email p o ec ed]
b
Depa amen de Qu´
ımica F´
ısica i Anal´
ı ica, Uni e si a Jaume I, 12071 Cas ell´
o,
Spain. E-mail: moline @uji.es
c
Dep o. de Qu´
ımica O g´
anica, Facul ad de Ciencias, Uni e si y o Za agoza, Ped o
Ce buna, 12, 50009, Za agoza, Spain
d
He e ogeneous Bioca alysis Labo a o y, Cen e o Coope a i e Resea ch in
Bioma e ials (CIC biomaGUNE), Basque Resea ch and Technology Alliance (BRTA),
Paseo de Mi am´
on 182, 20014, Donos ia San Sebas i´
an, Spain
e
IKERBASQUE, Basque Founda ion o Science, Ma ´
ıaD
´
ıaz de Ha o 3, 48013 Bilbao,
Spain
†We wan o dedica e his wo k o he memo y o P o . Jos´
e Ignacio Ga c´
ıa who
ge mina ed his wo k, encou aged us o ace his challenge and ac ed as
a p iceless ad iso , p o iding answe s and cons an ly suppo ing us. Res in
peace.
‡Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI:
10.1039/d0sc04471g
Ci e his: Chem. Sci., 2020, 11, 12009
All publica ion cha ges o his a icle
ha e been paid o by he Royal Socie y
o Chemis y
Recei ed 14 h Augus 2020
Accep ed 5 h Oc obe 2020
DOI: 10.1039/d0sc04471g
sc.li/chemical-science
This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12009
Chemical
Science
EDGE ARTICLE
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
View Jou nal
| View Issue
e al. epo ed a s aigh o wa d me hodology o syn hesise
glyce yl monoe he s om glycidol and se e al alcohols using
KOH as ca alys (Scheme 1B).
18
Ne e heless, he eac ion yields
a acemic p oduc ha makes he isola ion o he a ge enan-
iome a non- iable ask o p ac ical pu poses. To access one o
he enan iome s, a kine ic esolu ion is demanded. In his
con ex , lipases
19,20
a e efficien ca alys s o selec i ely ace yla e
only one enan iome o glyce ol de i a i es, so he un eac i e
enan iome is easily pu ied. Al hough alcohol dehyd ogenases
ha e also been applied o kine ic esolu ion o sec-alcohols,
21
hey ha e ne e been applied wi h glyce ol de i a i es. The
enan ioselec i e oxida ion o monoalkyl glyce ols will p o ide
a double oppo uni y o access enan iome ically pu e alkyl
glyce ols as building blocks o d ug syn hesis and manu ac u e
o 3-alkoxy/a yloxy-1-hyd oxyace one as a new amily o bio-
based sol en s o be explo ed (Scheme 1B).
8
Mo eo e , enzy-
ma ic syn hesis o hese hyd oxyace one monoe he s is
unp eceden ed bu highly in e es ing as key building blocks o
he biosyn hesis o non-na u al deoxysuga s
22
and cosme ics.
23
The bes candida es o ca alyse such selec i e oxida ion
would be he glyce ol dehyd ogenases (GlyDH) due o hei
na u al subs a e specici y. Bio echnological applica ions o
hese enzymes howe e a e mainly limi ed o he whole cell
p oduc ion o dihyd oxyace one
24
and aluable diols h ough
me abolic enginee ing.
25
Besides, his amily o enzymes has
also been widely applied in biosensing as he ac i e phase o
glyce ol senso s.
26
Ne e heless, hei subs a e scope is
ex emely na ow, es ic ed mainly o glyce ol in he oxida ion
di ec ion. Among he ew excep ions, he wild- ype GlyDHs om
Klebsiella genus a e able o oxidise a subs a e bulkie han
glyce ol, and ha p omiscui y inc eases when eplacing he
ca aly ic Zn
2+
by o he di alen me als.
27
Rema kably, he GlyDH
om En e obac e (Klebsiella)ae ogenes oxidises he R-phenyl-
glycol o 2-hyd oxyace ophenone in a egio- and enan iose-
lec i e manne .
28
Hund eds o examples endo se he imp o emen o enzyme
p ope ies (ac i i y, s abili y and selec i i y) and he widening o
hei subs a e scope h ough p o ein enginee ing. As sa e pa h
o enzyme op imiza ion, he scien ic communi y exploi s,
ei he sepa a ely o syne gis ically, andom and ex ensi e
mu agenesis campaigns h ough di ec ed e olu ion
29
and
minimalis mu a ions h ough si e-di ec ed mu agenesis
guided by s uc u al knowledge.
22,30,31
In he pa icula case o
GlyDH, one o he ew documen ed successes is he combina-
ion o DNA-shuffling and si e-di ec ed mu agenesis o expand
he GlyDH subs a e scope o o he diols. This a ian was 2.6
imes mo e ac i e owa ds 1,3-bu anediol han he wild- ype.
32
Despi e hese effo s, GlyDHs ha e ne e been challenged
agains bulkie subs a es like glyce ol monoe he s.
27
Besides e-shaping he ac i e si e o enzymes o imp o e
hei in insic unc ional ea u es, he enginee ing jou ney
mus include he enzyme immobilisa ion o ab ica e obus
bioca alys s ha a e easily o sepa a e om he eac ion media
and eadily o in eg a e in o diffe en bio eac o congu a ions.
In he las yea s, he immobiliza ion o enginee ed enzyme
a ian s has allowed hei applica ion unde indus ially ele-
an condi ions.
16,33–35
To ha aim, selec i e immobiliza ion
p o ocols ha assu e he ac i i y and inc ease he s abili y o he
enzymes upon he a achmen o he solid ca ie a e p e ended.
In his wo k, we a ionally enginee GlyDH om Bacillus
s ea o he mophilus (W -BsGlyDH) o selec i ely oxidise alkyl/a yl
glyce ols o 3-alkoxy/a yloxy-1-hyd oxyace ones. Be o e s a ing
he enginee ing campaign, we shined ligh on he unce ain ies
ound in i s ca aly ic mechanism. Besides explaining how his
enzyme posi ions he subs a e in o i s ac i e si e, Quan um
Mechanics/Molecula Mechanics (QM/MM) compu a ional
s udies also e eal i s enan iop e e ence owa ds he S-isome o
he alkyla ed glyce ol. This in o ma ion was ins umen al o
u he design mu a ions a he ac i e si e ha allowed he
enzyme accommoda ing bulkie glyce ol monoe he s. F om he
molecula design, we walked owa ds he p ocess de elopmen
by immobilizing he mos ac i e enginee ed a ian . The
esul ing he e ogeneous bioca alys eases he p oduc sepa a-
ion and inc eases he enzyme ope a ional s abili y. Finally, he
he e ogeneous bioca alys he ein p esen ed was coupled o
a one-po sequen ial chemo-enzyma ic p ocess o syn hesise
enan iopu e glyce ol monoe he s om glycidol and e hanol
wi hou in e media e pu ica ion s eps (Scheme 1C).
Ma e ials and me hods
Chemicals
Glyce ol, hyd ogen pe oxide, dihyd oxyace one (DHA), a in-
adenine-dinucleo ide sodium sal (FAD
+
), ace ic anhyd ide
Scheme 1 (A) D ugs con aining alkyl/a yl-glyce ol scaffolds (in blue).
(B) Re osyn hesis pa hway owa ds enan iopu e alkyl/a yl glyce yl
monoe he s and subs i u ed hyd oxyke ones om enewable s a ing
ma e ials. (C). Two-s ep chemo-enzyma ic ou e o access (R)-3-
(alkoxy/a yloxy)p opan-1,2-diol and hei co esponding 3-alkoxy/
a yloxy-1-hyd oxyace one.
12010 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020
Chemical Science Edge A icle
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
and N-me hylimidazole, kanamycin sul a e om S ep omyces
kanamyce icus and ampicillin, ca alase om bo ine li e (Hl-
CAT) we e acqui ed om Sigma-Ald ich (S . Louis, IL, USA).
Nico inamide adenine dinucleo ide educed disodium sal
(NADH), nico inamide adenine dinucleo ide sodium sal
(NAD
+
) and isop opyl b-D- hiogalac opy anoside (IPTG), we e
pu chased om GERBU Bio echnik GmbH (Wieblingen, Ge -
many). Glyce yl monoe he s and die he s we e syn hesised as
p e iously desc ibed (Table S1‡).
18,36
Cobal -ac i a ed aga ose
mic obeads 4BCL (AG-Co
2+
) (pa icle size; 50–15 mm, po e size;
112 nm and 15 mmol o Co
2+
pe g ca ie ) we e pu chased om
ABT echnologies (Mad id, Spain). P ecision plus p o ein™
s anda ds, mic o Bio-spin™ch oma og aphic columns and
B ad o d eagen we e acqui ed om BIORAD. All o he
eagen s and sol en s we e analy ical g ade o supe io .
Me hods
Si e-di ec ed mu agenesis o c ea e he a ian s o GlyDH. A
si e-di ec ed mu agenesis p o ocol was used o cons uc se en
GlyDH mu an s ( ee single mu an s, V131A, Y142A, L252A;
h ee double mu an s, V131A/L252A, V131A/Y142A, Y142A/
L252A; and one iple mu an , V131A/Y142A/L252A). These
mu an s we e cons uc ed by using he W -GlyDH gene as
empla e. The gene ic cons uc pET28b(+)_his-glydh was
de eloped in a p e ious wo k.
37
B iey, o in oduce he desi ed
amino acid change, he co esponding pai s o oligonucleo ides
(Table S2‡) we e used as p ime s in he polyme ase chain
eac ion (PCR) using he abo e men ioned cons uc as
empla e and high deli y NZYP oo DNA polyme ase. The
p oduc o he PCR was diges ed wi h DpnI ha exclusi ely
es ic s me hyla ed DNA. E. coli DH5acells we e ans o med
di ec ly wi h he diges ion p oduc . The plasmids ha ha bou
he mu a ed GlyDH genes we e iden ied by sequencing and
hen ans o med in o E. coli BL21(DE3) cells o exp ess he
co esponding enzyme a ian s.
Cloning o his- agged wa e o ming NADH oxidase a ian s
om Lac obacillus pen osus (Lp-NOX). The gene desc ibed by
Nowak e al.
38
was op imised o E. coli codon usage and syn-
hesised by Gensc ip Bio ech (Pisca away, NJ, USA). The
syn he ic gen was cloned in o pET28b(+) using NdeI and XhoI
es ic ion si es. DNA isola ion, plasmid pu ica ion, es ic ion
analysis, plasmid cons uc ion and DNA sequencing we e
ca ied ou by s anda d me hods.
39
Bac e ial s ains and g ow h condi ions. Glyce ol dehyd o-
genase om Bacillus S ea o he mophilus (BsGlyDH), NADH
oxidases om The mus he mophilus (T -NOX) and Lac obacillus
pen osus (Lp-NOX) we e o e exp essed in E. coli BL21 cells as
p e iously epo ed.
37,40
B iey, 1 mL o an o e nigh cul u e o
E. coli BL21(DE3) ha bo ing each plasmid was used o inocula e
50 mL o Lu ia-Be ain (LB) medium con aining he co e-
sponding an ibio ic; kanamycin (nal concen a ion 30 mg
mL
1
) was used o all enzymes bu T -NOX ha equi ed
ampicillin (nal concen a ion 50 mgmL
1
). The esul ing
cul u e was ae obically incuba ed a 37 C wi h o bi al shaking
a 250 pm un il he OD 600 nm eached 0.6. Ae wa ds, he
cul u e was induced wi h 1 mM IPTG. All enzymes we e induced
o 3 h a 37 C bu Lp-NOX was incuba ed a 21 C o 18 h. Ae
he induc ion ime, cells we e ha es ed by cen i uga ion a
4211 g o 30 min a 4 C. Supe na an s we e disca ded and he
pelle was esuspended in 5 mL o 25 mM sodium phospha e
buffe solu ion a pH 7. Cells we e b oken by sonica ion using
a Sonoplus Se ie 4200, Bandelin a 20% ampli ude (5 s ON/5 s
OFF) o 20 min a 4 C. The suspension was hen cen i uged a
10 528 g o 30 min a 4 C and he pelle was disca ded. The
supe na an con aining cell ex ac s wi h he His- agged
p o eins (excep T -NOX) we e collec ed and employed o
u he pu ica ion and/o immobiliza ion.
Pu ica ion o enzymes. The pu ica ion o GlyDH and he
a ian s, as well as he Lp-NOX we e done acco ding he
ollowing p ocedu e: 10 olumes o c ude cell ex ac con aining
he his- agged enzymes we e mixed wi h 1 olume o AG-Co
2+
mic obeads and incuba ed unde o bi al shaking o 1 o 2 h a
4C. La e , he suspension was l e ed and he mic obeads
con aining he enzyme we e washed wi h 5 olumes o 25 mM
phospha e buffe a pH 7. Ae wa ds, enzymes we e elu ed by
he addi ion o 5 olumes o 300 mM imidazole in sodium
phospha e buffe 25 mM pH 7 and incuba ed o 1 h a 4 C wi h
o bi al shaking. The elu ed p o ein was subjec ed o dialysis o
emo e he imidazole. Finally, SDS-PAGE and B ad o d p o ein
assay
41
we e ca ied ou ae each p oduc ion o de e mine he
pu i y, concen a ion and specic ac i i y o he enzymes
(Fig. S1‡). T -NOX was pu ied as p e iously desc ibed h ough
he mal shock.
40
Enzyma ic ac i i y measu emen s. Enzyme ac i i ies we e
spec opho ome ically measu ed in anspa en 96-well
mic opla es, employing a Mic opla e Reade Epoch 2, BioTek®
wi h he sowa e Gen5.
GlyDH ac i i y (colo ime ic assay). (1a, ac-1b, ac-1c, ac-1d,
ac-1e, ac-1 , ac-1g, ac-1h,1b-bis and 1c-bis) and 1 mM NAD
+
in sodium phospha e o sodium bica bona e buffe 100 mM a
pH 7 o 9, espec i ely; we e incuba ed wi h 5 mL o enzyma ic
solu ion o suspension a 30 C. The inc ease o he abso bance
was moni o ed a 340 nm. One uni o ac i i y was dened as he
amoun o enzyme ha was equi ed o educe 1 mmol o NAD
+
pe minu e a he assayed condi ions.
NOX ac i i y (colo ime ic assay). 200 mL o a eac ion mix u e
con aining 0.2 mM NADH and 150 mM FAD
+
in phospha e
buffe pH 7 we e incuba ed wi h 5 mL o enzyma ic solu ion o
suspension a 30 C. The dec ease in he abso bance was
moni o ed a 340 nm. One uni o ac i i y was dened as he
amoun o enzyme ha was equi ed o oxidise 1 mmol o NADH
pe minu e a he assayed condi ions.
CAT ac i i y (colo ime ic assay). The ac i i y was de e mined
by eco ding he dec ease in he abso bance a 240 nm o 200 mL
o a eac ion mix u e con aining 35 mM hyd ogen pe oxide in
sodium phospha e buffe 100 mM pH 7 a 30 C. The eac ion
was ini ia ed by adding 5 mL o he enzyma ic solu ion o
suspension o he eac ion mix u e. One uni o CAT ac i i y was
dened as he amoun o enzyme equi ed o he disp opo -
iona ion o one mmol o hyd ogen pe oxide pe minu e a he
assessed condi ions.
Co-immobiliza ion o Lp-NOX and BsGlyDH-L252A on AG-
Co
2+
.The enzymes we e immobilised by mixing 10 olumes o
This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12011
Edge A icle Chemical Science
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
c ude cell ex ac con aining he His- agged p o ein wi h 1
olume o AG-Co
2+
mic obeads and incuba ed unde o bi al
shaking o 1 o 2 h a 4 C. La e , he suspension was l e ed
and he mic obeads con aining he enzyme we e washed wi h 5
olumes o 25 mM phospha e buffe a pH 7. The immobilised
bioca alys was s o ed a 4 C. The co-immobilised bioca alys
comp ising GlyDH-L252A and Lp-NOX whe e p epa ed
ollowing he o me me hodology bu sequen ially immobiliz-
ing one by one enzyme, s a ing by he GlyDH-L252A ollowed by
he Lp-NOX.
Oxida ion o glyce yl e he s by GlyDH-L252A bioca alys s.
Fo ac-1c, immobilised GlyDH-L252A on AG-Co
2+
, T -NOX and
Hl-CAT we e mixed wi h 0.3–50 mL o 25–100 mM subs a e, as
indica ed, 1 mM NAD
+
, 150 mM FAD
+
in 100 mM sodium
phospha e buffe a pH 7. Fo ac-1h, co-immobilised GlyDH-
L252A and Lp-NOX on AG-Co
2+
was mixed wi h 1 mL o 2 mM
subs a e, 4 mM NAD
+
in 100 mM sodium phospha e buffe a
pH 7. Bo h mix u es we e incuba ed a 30 C in a e ical
o a ing shake a 40 pm. Reac ion cu se was moni o ed by
wi hd awing samples a pe iodic in e als ha we e analysed by
ch oma og aphic me hods.
Chemo-enzyma ic syn hesis. 0.87 mmol o KOH we e sol ed
in 3.81 mL o d ied absolu e e hanol (15 : 1 mol a io in espec
o glycidol). Then he mix u e was hea ed a 65 C and 0.3 mL o
ac-glycidol (4.35 mmol) we e added d opwise o 15 min. Ae
2 hou s, when ac-glycidol has comple ely been con e ed o ac-
1c, he empe a u e was cooled down o 25 C, KOH neu alised
wi h 0.3 M HCl and he sal s l e ed off.
He e ogeneous bioca alys GlyDH-L252A co-immobilised
wi h Lp-NOX on AG-Co
2+
(100 mg bioca alys ) was placed
inside a 1.5 mL Bio-spin™ch oma og aphic column. Then, 500
mL o eac ion mix u e consis ed in 20 mM o he ac-1c
subs a e (pu e, aw om chemosyn hesis wi h e hanol and aw
om chemosyn hesis wi hou e hanol), 1 mM NAD
+
, 150 mM
FAD
+
in 100 mM sodium phospha e buffe a pH 8 and we e
added and incuba ed a 30 C in a e ical o a ing shake a
40 pm. Ae 24 and 48 h, columns we e spin down and he
eco e ed eac ion mix u es we e analysed as desc ibed in he
ch oma og aphic me hods.
Ope a ional s abili y o bioca alys . Recycling o he e oge-
neous bioca alys BsGlyDH-L252A immobilised on AG-Co
2+
(144 mU, 100 mg bioca alys ) was de e mined by placing he
bioca alys inside a 1.5 mL Bio-spin™ch oma og aphic column
wi h 300 mL o eac ion mix u e consis ed in 25 mM o ac-1c,
1 mM NAD
+
, 150 mM FAD
+
, 720 mU o soluble T -NOX and
36 000 mU o Hl-CAT in 100 mM sodium phospha e buffe a
pH 7 and incuba ed a 30 C in a e ical o a ing shake a
40 pm. Ae each eac ion cycle (24 h), columns we e spin
down and washed one ime wi h one olume o 25 mM sodium
phospha e buffe a pH 7 be o e s a ing he nex eac ion cycle.
Reco e ed eac ion mix u es we e analysed as desc ibed in he
ch oma og aphic me hods.
Ch oma og aphic me hods
Gas ch oma og aphy (GC). P io GC analysis, samples we e
de i a ised as desc ibed elsewhe e.
42
B iey, 30 mL o aqueous
eac ion simple we e placed in a 1.5 mL Eppendo ube, ol-
lowed by he addi ion o 30 mLo N-me hylimidazole and 225 mL
o ace ic anhyd ide and incuba ed by 10 min a oom empe -
a u e. Ae wa ds, 300 mL o dis illed wa e was added and
allowed o cool down. La e , liquid–liquid ex ac ion o ace y-
la ed compounds was done by he addi ion o 300 mLo
dichlo ome hane con aining 2 mM eicosane as ex e nal s an-
da d disca ding he aqueous phase. 30–50 mg o anhyd ous
MgSO
4
we e added o d y samples be o e GC analysis. Gas
ch oma og aphy analyses we e ca ied ou in a Hewle Packa d
7890 Se ies II gas ch oma og aph using a column o phenyl
silicone 5.5% (Zeb on ZB-5HT In e no 30 m 0.25 mm 0.25
mm), helium as ca ie gas, and equipped wi h a ame ioniza-
ion de ec o (FID). Injec o a 250 C, FID a 280 C. Sepa a ion
o 1c de i a i es we e done by he ollowing empe a u e
p og am: ini ial empe a u e a 60 C, main ained 2 min, amp
o 240 C a a a e o 20 C min
1
and nally main ained 2 min.
Re en ion imes o ace yla ed compounds we e: ac-1c:7.88min,
3-e hoxy-1-hyd oxyace one: 6.75 min and eicosane: 12.2 min. The
samples we e addi ionally analysed using a Hewle Packa d 6890
Se ies II Gas ch oma og aph using a column o phenyl silicone
5.5% (Zeb on ZB-5HT In e no 30 m 0.25 mm 0.25 mm),
coupled o an Agilen 5973 ine Mass Spec ome e wi h Elec-
onic Impac ioniza ion.
Chi al high pe o mance liquid ch oma og aphy (HPLC).
Be o e hei analysis, samples we e l e ed and dilu ed in iso-
p opanol as equi ed. P epa ed samples we e analysed by HPLC
(Wa e s 2690) wi h a Lux 5 mcellulose-1 chi al column (250 mm 
4.60 mm) Phenomenex, equipped wi h a PDA de ec o a 270 nm.
Analy es we e elu ed a 1 mL min
1
cons an ow wi h a no mal
mobile phase composed by hexane/isop opanol (85 : 15).
43
Re en-
ion imes we e: R-1h: 11.9 min, S-1h:23.4minand1-phenoxy-3-
hyd oxyace one (2h): 5.4 min.
QM/MM compu a ional me hods. All he sys ems, wild- ype
BsGlyDH wi h glyce ol, S-1c and R-1c enan iome s, BsGlyDH-
L252A mu an wi h glyce ol, S-1c and R-1c enan iome s and
BsGlyDH-D123N mu an wi h glyce ol we e sol a ed by a box o
wa e molecules and we e neu alised adding coun e ions by
LEaP module
44
om Ambe Tools17 package.
45
P elimina y
minimiza ions and molecula mechanics (MM) and molecula
dynamics (MD) simula ions we e pe o med in o de o equili-
b a e he sys ems wi h he AMBER ff14SB and TIP3P o ce elds
o desc ibe he p o ein and wa e molecules, espec i ely.
Explo a ion o he QM/MM po en ial ene gy su aces (PESs),
and localiza ion and cha ac e iza ion o ansi ion s a e (TS)
s uc u es we e ca ied ou p io o he gene a ion o he QM/
MM ee ene gy su aces gene a ed in e ms o po en ials o
mean o ce (PMFs) a M06-2X(6-31+G(d,p)):PM3/AMBER/TIP3P
PMFs le el o heo y, including ze o poin ene gy (ZPE) co ec-
ions. Full compu a ional de ails a e gi en in he ESI.‡
Resul s and discussion
Enginee ing o GlyDH and mechanis ic insigh s
Inspi ed by p e ious wo ks,
46,47
we ske ched a e osyn he ic
pa hway o access enan iopu e alkyl/a yl glyce yl monoe he s
s a ing om inexpensi e epoxides such as glycidol, which can
be eadily syn hesized om glyce ol (Scheme 1B). Once acemic
glyce ol e he s a e chemically syn hesized unde basic
12012 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020
Chemical Science Edge A icle
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online

condi ions,
18
we p opose hei sequen ial enzyma ic egio- and
enan ioselec i e oxida ion. Un o una ely, none oxido educ ase
has been desc ibed o efficien ly conduc he selec i e oxida ion
owa ds his ype o monoglyce yl e he s. GlyDH is one o he
po en ial enzymes o ca alyse ha eac ion, since i efficien ly
oxidises glyce ol o dihyd oxyace one.
48
Ne e heless, i s oxida-
ion ac i i y is p ac ically es ic ed o i s na u al subs a e:
glyce ol. To go beyond glyce ol, we es ed wo GlyDHs om
diffe en bac e ial sou ces o he selec i e oxida ion o mono-
alkylglyce ols. We de ec ed some oxida i e ac i i y owa ds
a ba e y o pu e monoalkylglyce yl e he s using bo h wild- ype
GlyDH om Cellulomonas sp. (CsGlyDH) and om Bacillus
s ea o he mophilus (BsGlyDH) (Table S3‡). These esul s ag ee
wi h he esidual ac i i y p e iously epo ed o CsGlyDH
owa ds 1b.
49
Rema kably, he ela i e ac i i y o he enzyme
om Bacillus was highe han he one om Cellulomonas
owa ds a wide scope o glyce ol e he s. These insigh s mo i-
a ed us o enginee BsGlyDH h ough a s uc u e-guided
app oach.
The epo ed c ys al s uc u es (PDB ID: 1JQ5 and 1JQA)
48
o
BsGlyDH show ha O1 and O2 a oms o glyce ol a e coo di-
na ed o he ca aly ic Zn
2+
a om, which explains he exquisi e
specici y o his enzyme o 1,2-diols as subs a es. In con as ,
he O3 seems o be neu al in he ca aly ic mechanism bu i is
conned in o he ca i y o med by V131, Y142 and L252, sup-
po ing he ac , ha glyce ol is he p e e en ial subs a e
(Fig. 1). Despi e he a ailable s uc u al in o ma ion, he p ecise
ca aly ic mechanism o hyd ide ans e om C2 a om o
glyce ol o C4 a om in NAD
+
emains unclea (Fig. 2). Then,
be o e sculp ing he ac i e si e o accommoda e bulkie
subs i uen s in O3, we decided o explo e he ee ene gy
p oles o un eil he BsGlyDH mechanism a a omis ic le el
(see ESI‡). In he li e a u e, a wa e molecule is sugges ed o ac as
a base o abs ac he p o on om he C2 hyd oxyl g oup and o m
he alkoxide in e media e o subsequen ly p oceed wi h he
hyd ide ans e om C2 a om o glyce ol o NAD
+
co ac o .
48
Howe e , his mechanism has been demons a ed nei he h ough
expe imen al no compu a ional e idences. The e a e o he sho -
chain alcohol dehyd ogenases/ educ ases (SDRs) om D osophila
melanogas e and D osophila lebanonensis
50
ha con ain highly
conse ed ac i e si e esidues (Se and Ty ) ha ac as s ong base
o he p o on abs ac ion. Mo eo e , in manni ol 2-dehyd ogenase
om Pseudomonas uo escens,
51
a Lys esidue unc ions as he
ca aly ic base ha acili a es hyd ide ans e o NAD
+
by p o on
abs ac ion om alcohol. Classical MD simula ions o he ully
sol a ed subs a e–enzyme complex keep he Zn
2+
ion  e coo di-
na e complex, as in he ini ial X- ay s uc u e o Ruzheiniko e al.,
48
including he coo dina ion o O2 a om. Ne e heless, wo diffe en
con o ma ions o glyce ol bound o he ac i e cen e a e iden ied
(Fig. 2 and S2–S4‡). In one con o ma ion he p o on om O2 a om
o glyce ol es ablishes hyd ogen bond in e ac ion wi h a wa e
molecule while in he o he con o ma ion; his p o on es ablishes
a hyd ogen bond wi h D123 esidue. This ac led us o p opose wo
diffe en eac ion mechanisms (Fig. 2). Mechanism 1 in ol es he
p o on ans e om O2 a om o glyce ol o a wa e molecule ol-
lowed by a hyd ide ans e om C2 a om o glyce ol o C4 a om o
NAD
+
as p oposed in he li e a u e.
48
Con a iwise, mechanism 2
leads o a p o on ans e be ween he subs a e and he acid
esidue be o e he hyd ide ans e om glyce ol o NAD
+
.To
deciphe he mos likely mechanism, we gene a ed he ull ee
ene gy landscapes o he wo p oposed mechanisms by means o
mul iscale QM/MM simula ions. In pa icula , ee ene gy su aces
we e compu ed o e e y chemical s ep as po en ials o mean o ce
(PMFs) a M06-2X/6-31+G(d,p):PM3/MM le el o heo y (see ESI
Fig. S5 and S6‡).
Fig. 1 O e lap o c ys al s uc u es o GlyDH om Bacillus s ea -
o he mophilus binding i s na i e subs a e glyce ol (pink s icks/PDB ID:
1JQA), he ca aly ic Zn
2+
(blue sphe e/PDB ID: 1JQ5) and he NAD
+
(yellow s icks/PDB ID: 1JQ5). Residues V131, Y142 and L252 ha o m
he subs a e ca i y a e highligh ed as g een sphe es. Black dash line
ep esen s he hyd ide ans e om C2 a om o glyce ol o C4 a om o
NAD
+
(see Fig. 2). Yellow dash lines ep esen he coo dina ion o O1
and O2 a oms om glyce ol wi h he Zn
2+
a om.
Fig. 2 P oposed glyce ol oxida ion mechanisms o W -BsGlyDH. (1)
P o on ans e h ough wa e molecule. (2) P o on ans e h ough
he Asp123. R: glyce ol/TS: ansi ion s a e o mechanism 1/TS1 and
TS2 ansi ion s a e o mechanism 2/I1: alkoxide in e media e/P:
dihyd oxyace one.
This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12013
Edge A icle Chemical Science
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
Acco ding o he esul s, mechanism 1 can be disca ded due
o he esul ing o e all high ee ene gy ba ie
(57.6 kcal mol
1
), indica ing ha wa e is unable o ac as base
o he p o on sub ac ion om he O2 hyd oxyl g oup ( o
u he de ails see ESI, Fig. S5 and S6A‡). Fu he mo e, ou
M06-2X/6-31+G(d,p):PM3/MM ee ene gy su aces and M06-2X/
6-31+G(d,p)/MM po en ial ene gy su aces con ms ha
mechanism 2 akes place in a s epwise manne (Fig. S5 and
S6B‡), consis ing in he p o on abs ac ion o he alcohol by
D123 esidue ollowed by he hyd ide ans e om glyce ol o
NAD
+
. This second s ep, ha is he a e-limi ing one, ende s an
ac i a ion ee ene gy (17.1 kcal mol
1
) ha ag ees wi h he
alues ha can be de i ed om he expe imen al a e cons an s
(16.9 kcal mol
1
).§To expe imen ally con m he mechanism 2,
we cons uc ed he mu an D123N ha keeps he hyd ogen
bond in e ac ions be ween glyce ol and he amide g oup o
D123N wi hou affec ing he size bu p ecluding he o ma ion
o he alkoxy in e media e, as he amide g oup is unable o
sub ac he p o on. D123N mu an indeed abolished he
oxida i e ac i i y (Fig. S7‡), which ag ees wi h he analysis o he
geome ies de i ed om classical MD simula ions ha show no
al e na i e base in he ac i e si e. Hence, we p opose he
ca boxyla e o D123 as he base needed o abs ac ing he
p o on o o m he alkoxy in e media e ha p ecedes he
hyd ide ans e o he nico inamide co ac o (Fig. 2). Fo he
 s ime, we p esen bo h expe imen al and compu a ional
da a ha unambiguously un eil he key ole o D123 in he
ca aly ic mechanism o a glyce ol dehyd ogenase (Fig. 2,
mechanism 2, Fig. S5‡). This esidue is highly conse ed in
GlyDHs and glyce ol 1-phospha e dehyd ogenases (Fig. S8 and
S9‡), which suppo s i s impo an ole in he ca aly ic mecha-
nism. Unlike manni ol dehyd ogenases
52
(medium chain
dehyd ogenases) and sho -chain dehyd ogenases,
50
whe e Lys
and Ty esidues ac s as gene al base, espec i ely, BsGlyDH
uses he ca boxyla e o D123 o sub ac he p o on om he
seconda y alcohol o glyce ol.
53–56
This conse ed esidue is no
ound in i on-con aining polyol dehyd ogenases, like 1,2-p op-
anodiol dehyd ogenase.
57
Once he oxida ion mechanism has been elucida ed, we
a ionally selec ed h ee posi ions o sculp he ac i e si e o
BsGlyDH o be e accommoda e bulkie glyce ol de i a i es.
We pe o med an alanine scanning in he posi ions V131, Y142
and L252 since hey s e ically conne he C3 hyd oxyl g oup
wi hin he enzyme binding pocke acco ding o he X- ay
s uc u e (Fig. 1). We also cons uc ed he h ee possible
combina ions o double mu an s and he iple one. By eplac-
ing hese bulkie amino acids by alanine, we we e able o
expand he sol en accessible a ea o he ac i e si e up o 108 ˚
A
2
(Table S4‡). Ne e heless, he e was no co ela ion be ween he
size o he binding pocke and he ac i i y owa ds he glyce yl
e he s (Fig. S10‡). All mu a ions d ama ically educed he
enzyme ac i i y (<10%) owa ds he na i e subs a e 1a (glyce ol)
excep BsGlyDH-V131A (Fig. 3). On he o he hand, he mu a ion
Y142A conduc ed o inac i e mu an s owa ds any es ed
subs a e ega dless hey we e single, double o iple a ian s.
Pleasan ly, he a ian BsGlyDH-L252A p esen ed a ema kable
ac i i y owa ds 1c, e en 30% highe han he oxida i e ac i i y
o he W -GlyDH owa ds glyce ol. Fo all hose ac i e mu an s,
we s udied hei subs a e scope using a pale e o alkyl/a yl
glyce yl monoe he s (Table S1‡). BsGlyDH-L252A was he mos
ac i e a ian owa ds all alkyla ed glyce ol de i a i es, while
negligible ac i i y was de ec ed o he 1,3-dime hoxy (1b-bis)
and 1,3-die hoxy (1c-bis) glyce yl de i a i es. The absence o
ac i i y owa ds he glyce yl 1,3-dialkyle he s ag ees wi h he
ac ha GlyDH equi es one ee p ima y hyd oxyl g oup o be
coo dina ed wi h he ca aly ic Zn
2+
. In iguingly, widening he
ac i e si e did no enhance he oxida i e ac i i y owa ds a yl
de i a i es. In ac , we ound ou ha bo h wild- ype and he
L252A a ian we e simila ly ac i e owa ds 1h.
The second bes subs a e was 1g bea ing a 2,2,2- iuo e hyl
subs i uen , which suppo s ha he 22 ˚
A
2
la ge ac i e si e also
possesses he sui able shape o accommoda e CH
2
CF
3
moie ies
(Table S4‡). Su p isingly, when he alipha ic chain was ei he
smalle o la ge han 2 ca bons, he enzyme ac i i y d ama i-
cally decayed (Fig. 3). The specic ac i i y esul s we e sup-
po ed by kine ic s udies (Table 1, Fig. S11 and S12‡), which
addi ionally demons a e ha sho ening he apola side chain
a posi ion 252 d ama ically inc eases he K
M
owa ds glyce ol
wi hou signican ly affec ing he k
ca
. In con as , mu a ion
L252A signican ly inc eases (6 imes) he BsGlyDH-L252A
ca aly ic efficiency owa ds 1c due o he k
ca
enhancemen
(163 imes). This a ian showed simila ends owa ds all alkyl
glyce yl de i a i es excep 1e, o which he k
ca
was signican ly
lowe compa ed o o he subs i uen s (Table 1). BsGlyDH-L252A
p esen ed simila K
M
alues owa ds NAD
+
unde he same
condi ions using bo h glyce ol and 1c as subs a es, which
indica es ha he mu a ion clea ly affec s he binding o he
polyol a he han he co ac o (Fig. S13C–F‡). Using glyce ol as
subs a e a pH 7, we also obse ed ha bo h wild- ype and
mu an enzymes we e inhibi ed by NAD
+
a high concen a ion
(>10 mM) (Fig. S13A–D‡). In he specic case o BsGlyDH-
L252A, inhibi ion negligibly occu ed when using 1c as
subs a e unde neu al condi ions (Fig. S13E and F‡), bu i was
no iceable a pH 9 (Fig. S13G and H‡). Rema kably, he ca aly ic
efficiency o BsGlyDH-L252A was 40 imes highe unde alkaline
condi ions han unde pH 7, and i s op imal pH was shied 1
uni owa ds basic alues compa ed o he wild- ype enzyme
Fig. 3 Specific ac i i y o wild- ype and se e al mu an s o BsGlyDH
owa ds alkyl/acylglyce yl e he s. In all cases, eac ion mix u es con-
sis ed in 100 mM subs a e, 1 mM NAD
+
in 100 mM sodium phospha e
buffe a pH 7 a 30 C, 1b-bis and 1c-bis a e he co esponding dia-
lkyl-e he wi h me hyl and e hyl subs i uen s, espec i ely.
12014 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020
Chemical Science Edge A icle
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
(Fig. S14‡). These kine ics s udies e eal an impo an ole o
pH on he glyce ol dehyd ogenase pe o mance ha affec s
bo h binding (o e all o he co ac o ) and eac ion a e
cons an .
58
Fig. 4, S15 and S16‡show he ee ene gies p oles
and he hyd ide ans e ansi ion s a es (named TS2, see
Fig. 2) esul ed om QM/MM simula ions, o he oxida ion
eac ion o diffe en subs a es ca alysed by ei he he W -BsGlyDH
o he BsGlyDH-L252A mu an . Ene gy ba ie s ollow he same
end obse ed o he expe imen al kine ics shown in Table 1. This
mu an p esen s an ene gy ba ie o 16.2 and 16.5 kcal mol
1
o
bo h 1c (Fig. 4A) and glyce ol (Fig. S16‡) oxida ions, espec i ely.
These alues a e sligh ly smalle han he ba ie p e iously ob-
ained wi h he wild- ype enzyme using glyce ol as subs a e
(17.1 kcal mol
1
) (Fig. S5‡). Mo e in e es ingly, he ee ene gy
p ole o W -BsGlyDH shows an ene gy ba ie 2.6 kcal mol
1
highe han BsGlyDH-L252A owa ds he oxida ion o 1c unde he
same condi ions (Fig. 4A), in acco dance wi h he expe imen al ee
ene gy ba ie s de i ed om he k
ca
alues (Tables 1, S5‡). We
sugges ha he highe efficiency o enginee ed BsGlyDH-L252A
esul s om a less s e ically impeded 1c con o ma ion wi hin he
mu a ed ac i e si e, which leads o ansi ion s a es wi h lowe
ene gy ba ie s han wi hin na i e one. QM/MM MD simula ions
e ealed weak bu s abilizing in e ac ions ene gies be ween 1c and
he esidues a posi ions 131, 142 and 252 o BsGlyDH-L252A
du ing he eac an s a e. Thei syne gis ic Lenna d-Jonnes in e -
ac ions signican ly con ibu e o he 1c binding (Fig. S17 and
S18‡). The e o e, he inse ion o Ala a posi ion 252 bu keeping
he na i e esidues a posi ions V131 and Y142 seems o op imise
he binding pocke o imp o e he s abili y o he ansi ion s a es
wi h he e hyl glyce yl monoe he . Fig. 4B shows indeed a o a ion
(125 deg ees) o he CH
3
- o e hoxy subs i uen o 1c wi hin he less
hinde ed L252A ac i e si e. Simila esul s we e ound o he
a ional si e-di ec ed mu agenesis o a shuffled GlyDH, whe e he
eplacemen o one aspa ic ha clashed wi h 1,3-bu anodiol
enhanced he oxida i e ac i i y up o 2.6 imes compa ed o he
wild- ype a ian .
32
The excellen oxida i e ac i i y o BsGlyDH-L252A encou -
aged us o ca y ou he comple e biocon e sion o 1c and
cha ac e ise he esul ing p oduc . Since his bio ans o ma ion
is NAD
+
-dependen , we mixed BsGlyDH-L252A wi h an NADH
oxidase om The mus he mophilus HB27 (T -NOX) and he
ho se li e ca alase (Hl-CAT) in one-po , aiming a bo h in si u
ecycling he co ac o and emo ing he H
2
O
2
; a oxic by-
p oduc om he ecycling sys em. Using he soluble
enzymes, he eac ion oughly eached 25% subs a e con e -
sion in 72 hou s wi h enzyme and co ac o o al u no e
numbe s o 3491 and 13, espec i ely (Table 2, en y 1). Despi e
ha low con e sion, we cha ac e ised he o med p oduc
h ough
13
C and
1
H NMR (Fig. S19–S22‡), and mass spec om-
e y (Fig. S23–S29‡). The analyses con m ha BsGlyDH-L252A
oxidises he C2 o 1c as he wild- ype GlyDH does o he glyc-
e ol. Rema kably, we did no de ec he o ma ion o he alde-
hyde, which indica es ha he mu a ion a he ac i e si e al e s
he subs a e specici y bu no he enzyme egioselec i i y.
Al hough p oduc yields we e signican ly lowe han hose
ones ob ained h ough chemical me hods,
59
he egioselec i i y
o his a ian owa ds he C2 o he alkylglyce yl subs a e hus
enables he syn hesis o he co esponding 3-alkoxy-1-
hyd oxyace ones unde mild condi ions using wa e as
sol en . Mo e impo an ly, he enzyma ic ou e smoo hly uns
wi hou p o ec ion/dep o ec ion s eps unde acidic condi ions
ha would p o oke he decomposi ion o he p oduc o me hyl
glyoxal, subs an ially dec easing he isola ed yields.
59
Bio ans o ma ion o alkyl/a yl glyce yl e he s using
immobilised GlyDH-L252A
In o de o in ensi y he p ocess and walk owa ds highe yields,
we immobilised BsGlyDH-L252A on aga ose po ous mic obeads
ac i a ed wi h cobal -chela es (AG-Co
2+
) o enhance i s ope a ional
s abili y and inc ease he bioca alys olume ic ac i i y. Fu he -
mo e, he use o he immobilised enzyme will ease he p oduc
eco e y and enable he enzyme eu iliza ion once he eac ion is
comple ed. Since all BsGlyDH a ian s used in his wo k we e
agged wi h 6His polypep ide a hei N- e minus o pu ica-
ion pu poses, we ha nessed ha ag o si e-selec i ely immobilise
he BsGlyDH-L252A on AG-Co
2+
. This a ian was quan i a i ely
immobilisedon hisca ie ;howe e , hespecic ac i i y o he
immobilised enzyme was educed o 19% compa ed o i s ee
coun e pa unde colo ime ic assay condi ions (Table S6‡). The
immobilised BsGlyDH-L252A pe o med he selec i e oxida ion o
1c wi h a TOF 2.4- old highe han he soluble enzyme, eaching
amaximumyieldo 50%ae 72 h unde ba ch condi ions (Table
Table 1 Michaelis–Men en s eady-s a e pa ame e s o W -BsGlyDH and BsGlyDH-L25A on diffe en glyce yl e he s
a
Subs a e
K
M
(mM) V
max
(U mg
1
)k
ca
(s
1
)k
ca
/K
M
(M
1
s
1
)
W L252A W L252A W L252A W L252A
1a 50 3 1880 80 1.40 0.04 1.70 0.05 7.4 0.2 9.0 0.3 148 5
1b 45 4 977 131 0.160 0.003 1.25 0.06 0.84 0.02 6.6 0.3 19 7
1c 13.4 0.4 371 71 0.036 0.001 6.0 0.5 0.190 0.003 31 31485
1d 30 1 608 102 0.052 0.003 2.2 0.2 0.27 0.02 12 1919
1e n.a 162 24 n.a 0.24 0.02 n.a 1.3 0.1 n.a 8
1 31.2 0.3 707 77 0.057 0.001 2.4 0.1 0.300 0.001 13 11018
1g 78 8 281 24 0.06 0.02 2.8 0.1 0.32 0.01 14 1451
1h 72 n.a 0.14 0.01 n.a 0.7 0.1 n.a 112 n.a
a
Ac i i y assay: 1 mM NAD
+
in 100 mM sodium phospha e buffe pH 7 a 30 C. n.a: no assessed because i did no each he subs a e sa u a ion.
This jou nal is © The Royal Socie y o Chemis y 2020 Chem. Sci.,2020,11,12009–12020 | 12015
Edge A icle Chemical Science
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online
2, en y 2). This he e ogeneous bioca alys enabled an enzyme and
co ac o o al u no e numbe s o 64 10
3
and 25, espec i ely,
unde he s udied condi ions.
These da a demons a e ha immobilised BsGlyDH-L252A
mixed wi h soluble T -NOX and Hl-CAT ou pe o ms he
sys em wi h he h ee soluble enzymes. The appa en lowe
ac i i y o he immobilised enzyme seems o be compensa ed by
i s highe obus ness unde ope a ional condi ions.
60
The
immobiliza ion o BsGlyDH-L252A allowed us inc easing 8
imes he olume ic ac i i y o he he e ogeneous bioca alys ,
eaching 50% con e sion in only 24 h (Table 2, en y 3).
F om he eac ion ime cou ses, we obse ed how he
oxida ion eac ion slows down when he con e sion app oaches
o 50% (Fig. 5A). Since we used 25 mM acemic mix u e o 1c,
ha asymp o ic beha io poin s ou ha BsGlyDH-L252A is
ex ao dina ily selec i e o one o he wo enan iome s. To
un eil he enan ioselec i i y o his a ian , we pe o med
u he QM/MM compu a ional s udies on he wo isome s o
1c. The ee ene gy ba ie s o he oxida ion o S-1c
(16.16 kcal mol
1
) was signican ly lowe han ha o he R-
enan iome (44.40 kcal mol
1
) (Fig. 4A), indica ing ha he
enginee ed BsGlyDH a ian p e e s oxidizing he S-enan-
iome . Simila esul s we e obse ed o he wild- ype enzyme
(Fig. S15 and Table S5‡), sugges ing ha he na i e ac i e si e
also p e e s he S-congu a ion o he subs a e o he hyd ide
ans e o he NAD
+
. Fig. 4C illus a es an unp oduc i e posi-
ion o R-1c as he seconda y alcohol loses i s coo dina ion wi h
he ca aly ic Zn
2+
. The same p edic ed enan iop e e ence o
bo h enginee ed and na i e a ian s sugges s ha he L252A
mu a ion aids he accommoda ion o la ge glyce ol-like
subs a es wi hou affec ing he egio- and s e eoselec i i y o
he enzyme. This S-enan iop e e ence o BsGlyDH ag ees wi h
he enan ioselec i i y epo ed o a cheal glyce ol-1-phospha e
dehyd ogenase,
61
sugges ing a p o-R posi ion o he NAD
+
wi hin he ac i e si e. GlyDHs om En e obac e ae ogenes and
om Cellulomonas sp. selec i ely oxidise he R-isome o 1,2-
diols, kine ically esol ing he co esponding S-enan iome s o
phenylglycol (ee 99%, con . 50%)
58
and 1,2-p opanodiol (ee
36%, con . 26%),
49
espec i ely. The opposi e CIP p io i y
be ween 1c and non alkoxyla ed 1,2-diols indica es ha he
diols a e simila ly coo dina ed and posi ioned wi hin he
enzyme ac i e si es o hose GlyDHs, and he hyd ide om he
subs a es is also ans e ed o Re- ace o he NAD
+
. Embold-
ened by hese compu a ional insigh s, we challenged he
immobilised BsGlyDH-L252A o he kine ic esolu ion o ac-1h
(Figs. S29–S33‡). Fig. 5B shows ha he eac ion ime cou se
ollows he classical kine ic esolu ion pa e n whe e he S-
enan iome is ully oxidised o 3-phenoxy-1-hyd oxyace one,
while he R-enan iome emains non eac i e (Fig. S33‡). This
esul con ms he compu a ional s udies and hus demon-
s a es ha BsGlyDH-L252A is an S-s e eoselec i e
polyol dehyd ogenase, en iching he eac ion c udes wi h R-1h
(ee > 99%) ae 72 hou s. Since we exploi ed BsGlyDHA-L252A
o selec i ely oxidise alkyl and a yl glyce yl monoe he s, we
a e able o eadily p epa e he pu e R-enan iome o glyce ol
monoe he s in one-po unde mild condi ions. To he bes o
ou knowledge, he only epo ed bio ans o ma ions able o
p oduce his enan iome is based on asymme ic educ ion o
hyd oxyace one monoe he s h ough whole cells o diffe en
yeas s, bu wi h signican ly lowe enan iopu i y (ee ¼73–
Fig. 4 (A) F ee ene gy p ofiles (M06-2X/6-31+G(d,p):PM3/MM PMFs +
ZPE co ec ion) ollowing mechanism 2 o he S-1c oxida ion ca a-
lysed by W -BsGlyDH (dashed o ange line) and BsGlyDH-L252A (solid
g een line), and o R-1c oxida ion ca alysed by BsGlyDH-L252A (solid
pu ple line). (B) O e lapped snapsho o he hyd ide ans e ansi ion
s a es (TS2) loca ed in he oxida ion o S-1c ca alysed by he W -
BsGlyDH (o ange) and BsGlyDH-L252A (g een). NAD
+
bound o he
complex o W and L252A mu an was colou ed in o ange and yellow,
espec i ely. (C) O e lapped snapsho s o he hyd ide ans e ansi-
ion s a e (TS2) loca ed in he oxida ion o S-1c (g een) and R-1c
(pu ple) by BsGlyDH-L252A. NAD
+
bound o he complex wi h Sand R
isome s was colou ed in yellow and pale g een, espec i ely.
12016 |Chem. Sci.,2020,11,12009–12020 This jou nal is © The Royal Socie y o Chemis y 2020
Chemical Science Edge A icle
Open Access A icle. Published on 05 Oc obe 2020. Downloaded on 3/8/2021 9:50:12 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion-NonComme cial 3.0 Unpo ed Licence.
View A icle Online