Inc eased The mos abili y o an Enginee ed Fla in-Con aining
Monooxygenase o Remedia e T ime hylamine in Fish P o ein
Hyd olysa es
Ma ianne Go is,aIsabel Cea-Rama,bPål Pun e oll,aRasmus Ree,aDa id Almend al,cJulia Sanz-Apa icio,bManuel Fe e ,c
G o Elin Kjæ eng Bje gaa
a
NORCE Clima e & En i onmen - NORCE No wegian Resea ch Cen e, Be gen, No way
b
Ins i u o de Quimica Fisica Rocasolano (IQFR), CSIC, Mad id, Spain
c
Ins i u o de Ca alisis y Pe oleoquimica (ICP), CSIC, Mad id, Spain
ABSTRACT P o ein hyd olysa es made om ma ine by-p oduc s a e e y nu i ious
bu equen ly con ain ime hylamine (TMA), which has an una ac i e fish-like smell.
Bac e ial ime hylamine monooxygenases can oxidize TMA in o he odo less ime hyl-
amine N-oxide (TMAO) and ha e been shown o educe TMA le els in a salmon p o ein
hyd olysa e. To make he fla in-con aining monooxygenase (FMO) Me hylophaga aminisul-
fidi o ans ime hylamine monooxygenase (mFMO) mo e sui able o indus ial applica ion,
we enginee ed i using he P o ein Repai One-S op Shop (PROSS) algo i hm. All se en
mu an a ian s, con aining 8 o 28 mu a ions, displayed inc eases in mel ing empe a u e
o be ween 4.7°C and 9.0°C. The c ys al s uc u e o he mos he mos able a ian ,
mFMO_20, e ealed he p esence o ou new s abilizing in e helical sal b idges, each
in ol ing a mu a ed esidue. Finally, mFMO_20 significan ly ou pe o med na i e mFMO
in i s abili y o educe TMA le els in a salmon p o ein hyd olysa e a indus ially ele an
empe a u es.
IMPORTANCE Ma ine by-p oduc s a e a high-quali y sou ce o pep ide ing edien s, bu
he unpleasan fishy odo caused by TMA limi s hei access o he ood ma ke . This
p oblem can be mi iga ed by enzyma ic con e sion o TMA in o he odo less TMAO.
Howe e , enzymes isola ed om na u e mus be adap ed o indus ial equi emen s,
such as he abili y o ole a e high empe a u es. This s udy has demons a ed ha mFMO
can be enginee ed o become mo e he mos able. Mo eo e , unlike he na i e enzyme, he
bes he mos able a ian e ficien ly oxidized TMA in a salmon p o ein hyd olysa e a indus-
ial empe a u es. Ou esul s p esen an impo an nex s ep owa d he applica ion o
his no el and highly p omising enzyme echnology in ma ine bio efine ies.
KEYWORDS fla in-con aining monooxygenases, ime hylamine, p o ein hyd olysa e,
enzyme enginee ing, PROSS
Fla in-con aining monooxygenases (FMOs, EC 1.14.13.8) a e enzymes ha inse one
molecule o oxygen in o o ganic subs a es using he co ac o s fla in adenine dinucleo ide
(FAD) and NAD(P)H (1–3). A subg oup o bac e ial FMOs oxidize ime hylamine (TMA) o i-
me hylamine N-oxide (TMAO) (Fig. 1) and a e o en e e ed o as ime hylamine monooxyge-
nases (Tmms) (4–6). These and o he FMOs ha e also gained in e es o hei abili y o con e
indole in o he dye indigo and he d ug agen indi ubin (5, 7–9). TMA is a well-known con ib-
u o o he odo o spoiled fish (10) and may accumula e o gi e ise o a s ong bodily
odo in humans wi h ime hylaminu ia (fish odo synd ome) caused by impai men s in
he FMO3 gene (11).
Fish p o ein hyd olysa es made om by-p oduc s om fishe ies and aquacul u e a e
o high nu i ional alue and ha e a g ea po en ial o he human consump ion ma ke
Edi o Ma ina Lo i, Uni e si y o Milano-
Bicocca
Copy igh © 2023 Go is e al. This is an open-
access a icle dis ibu ed unde he e ms o
he C ea i e Commons A ibu ion 4.0
In e na ional license.
Add ess co espondence o G o Elin Kjæ eng
Bje ga, [email p o ec ed].
The au ho s decla e no conflic o in e es .
Recei ed 10 Ma ch 2023
Accep ed 8 May 2023
Published 24 May 2023
June 2023 Volume 89 Issue 6 10.1128/aem.00390-23 1
ENZYMOLOGY AND PROTEIN ENGINEERING
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(12, 13). Howe e , fish p o ein hyd olysa es equen ly su e om an o -pu ing malodo
ha is mainly caused by TMA. Cu en ly, he TMA malodo may be handled by odo
masking, apo iza ion, encapsula ion, o fil a ion, albei wi h a ious deg ees o success
and possibly also comp omising o he quali ies in he p oduc s. The applica ion o Tmm
enzymes is hus an al e na i e and no el s a egy o con e TMA o he odo less TMAO in
fish p o ein hyd olysa es. This has he po en ial o significan ly imp o e he o ganolep ic
quali y o fish p o ein hyd olysa es and he eby p omo e hei applica ion as ood ing e-
dien s, while simul aneously main aining hei nu i ional p ofile.
In a p e ious s udy, we sc eened 45 bac e ial Tmms o hei abili y o oxidize TMA
o TMAO (6) and iden ified he Me hylophaga aminisulfidi o ans Tmm (mFMO) (5) as a sui a-
ble candida e o applica ion on a TMA-con aining salmon p o ein hyd olysa e. In indus ial
fish p o ein hyd olysis, enzymes a e equi ed o pe o m a pHs o a ound 6 and empe a-
u es anging om 45°C o 60°C (14, 15). This implies ha mFMO, wi h an op imal empe a-
u e o 44.0°C and mel ing empe a u e o 46.7°C, would benefi om enzyme enginee ing
o inc ease i s s abili y (6). In ha espec , a p e ious e o o enginee mFMO is encou ag-
ing. Lon
ca and colleagues used he compu a ional p o ocol FRESCO (16, 17) o p edic wo
mu a ions in mFMO, M15L (a mu a ion o M o L a posi ion 15) and S23A, ha when com-
bined inc eased he appa en mel ing empe a u e by 3.0°C (16).
The mFMO enzyme o ms a dime , and each monome consis s o wo domains: he
la ge FAD-binding domain and he smalle NADPH-binding domain (18, 19). Upon binding,
NADPH educes he igh ly bound FAD, hus gene a ing he eac i e fla in in e media e
C4a-hyd ope oxy-FAD and NADP
1
. The la e s abilizes he ac i a ed fla in in e media e,
and oge he wi h he y osine esidue a posi ion 207, i shields he ac i e si e and he in e -
media e om he sol en (18). When en e ing he ac i e si e, he subs a e displaces NADP
1
and is subsequen ly oxidized by he ac i a ed fla in in e media e.
P o ein Repai One-S op Shop (PROSS) is a Web se e ha akes a p o ein s uc u e as
inpu and ou pu s se e al mu a ed sequences ha a e expec ed o ha e inc eased s abili y
(20). PROSS combines mul iple independen ly s abilizing mu a ions by in eg a ing Rose a
modeling and phylogene ic sequence in o ma ion (20). In a ecen communi y-wide expe -
imen al e alua ion o PROSS, designs o 9 o 10 es ed p o ein a ge s displayed inc eases
in empe a u e s abili y ha anged om 8.3°C o 27.0°C (21).
In he p esen s udy, we employed he PROSS algo i hm on mFMO o imp o e i s he mal
s abili y. Se en combina o ial mu an a ian s o mFMO, con aining 8 o 28 mu a ions, we e
analyzed o hei empe a u e s abili y and compa ed o wild- ype mFMO. We demons a e
ha all mFMO a ian s we e mo e he mos able han he wild ype. The mos he mos able
a ian was analyzed by s eady s a e kine ics and compa ed o he wild ype wi hou iden i y-
ing subs an ial modifica ion o he kine ic pa ame e s. Mo eo e , his s abilized mFMO a ian
also con e ed TMA o TMAO mo e e ficien ly han na i e mFMO in a salmon p o ein hyd oly-
sa e a wo indus ially ele an empe a u es, 50.0°C and 65.0°C. Finally, he c ys al s uc u e
o he mos he mos able a ian was sol ed o elucida e he s uc u al basis o he inc eased
he mal s abili y, e ealing loss o flexibili y h ough a no el ne wo k o pola in e ac ions as
he main con ibu ing ac o .
RESULTS
Design and exp ession o mu an a ian s o mFMO wi h p edic ed inc eased
s abili y. To make a mo e s able and empe a u e- esis an mFMO, ideally wi hs anding
a leas 50°C in indus ial applica ions, we employed compu a ional enzyme enginee ing.
FIG 1 Chemical eac ion ca alyzed by ime hylamine monooxygenases.
The mos able T ime hylamine Monooxygenase Applied and En i onmen al Mic obiology
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The mos ecen c ys al s uc u e o mFMO in complex wi h he co ac o s FAD and NADP
1
(PDB iden ifica ion code [ID] 2XVH)(19) was used as inpu o he PROSS Web se e (20),
along wi h ins uc ions o exclude esidues in con ac wi h he co ac o s, as well as dime
in e ace esidues,asmu a ional a ge s.PROSSp oposed7mFMO a ian swi h henum-
be o mu a ions anging om 8 o 28 (Fig. S1 in he supplemen al ma e ial). The a ian s
we e named mFMO_n,whe enindica es he numbe o mu a ions. The mu a ions we e
loca ed a o nea he su ace, and he numbe o esidues p edic ed o o m new s abi-
lizing sal b idges inc eased om 2 in mFMO_8 o 8 in mFMO_28 (Table 1). In he models
o mFMO_8 h ough mFMO_20, all new sal b idges we e p edic ed o o m be ween one
mu a ed and one na i e esidue (Table 1). The las wo a ian s displayed mo e complex sal
b idge pa e ns, including di ec in e ac ions be ween mu a ed esidues: in mFMO_24, he
newly in oduced N394K mu a ion is p edic ed o o m sal b idges wi h bo h T370D and
D374, and in mFMO_28, he newly in oduced P391D mu an is p edic ed o o m a hi d
sal b idge wi h N394K (Table 1). In e es ingly, he majo i y o he new sal b idges a e p e-
dic ed o o m in e helical connec ions (Fig. 2, Table 2). The numbe o p edic ed sal b idges
ha connec seconda y s uc u e elemen s g adually inc eases om 1 in mFMO_8 o 6 in
mFMO_24 and mFMO_28.
All 7 mFMO mu an a ian s we e exp essed wi h a C- e minal hexahis idine ag, a
le els compa able o ha o na i e mFMO, pu ified (Fig. S2), and e ified by mass spec-
ome y. When exp essing mFMO in Esche ichia coli, he cul u e medium u ns blue
due o he enzyma ic con e sion o endogenous indole o indigo (5–7). The ac ha
he cul u e media o all mFMO a ian s u ned blue ollowing o e nigh exp ession
sugges ed ha he exp essed mFMO a ian s we e unc ional. Mo eo e , all pu ified
mFMO a ian enzymes we e colo ed b igh yellow, indica ing he p esence o bound
FAD co ac o , which is equi ed o unc ion.
mFMO mu an a ian s a e unc ional and mo e he mos able han na i e mFMO.
To in es iga e whe he he mFMO a ian s had inc eased he mal s abili y compa ed
o ha o he na i e enzyme, we conduc ed p o ein mel ing s udies using ci cula dich oism
(CD). The mel ing empe a u e (T
m
) o na i e mFMO was measu ed o be 46.2°C (Table 2),
which is in line wi h he p e iously epo ed T
m
o 46.7°C (6). All mFMO a ian s demon-
s a ed inc eased empe a u e s abili y compa ed o ha o he wild- ype enzyme, as
eflec ed by hei T
m
alues, which anged om 50.9°C o mFMO_28 o 55.2°C o mFMO_20
(Table 2). The mel ing empe a u e inc eased wi h he numbe o mu a ions om mFMO_8 o
mFMO_20 bu declined sligh ly o mFMO_24 and mFMO_28.
To s udy he inc eased empe a u e s abili y o he mFMO a ian s u he , we e al-
ua ed hei esidual ca aly ic ac i i y agains TMA a e 1-h incuba ions a empe a u es
om 30.0°C o 54.0°C. The empe a u e a which hal he enzyme ac i i y was los (T
50
)
anged om 45.1°C o mFMO_8 o 50.0°C o mFMO_20, all ou pe o ming na i e mFMO,
TABLE 1 P edic ed new sal b idges o med be ween mu a ed esidues and na i e o mu a ed esidues
Mu a ed
esidue
Residue o ming a p edic ed sal b idge in indica ed mu an mFMO a ian
a
mFMO_8 mFMO_11 mFMO_14 mFMO_15 mFMO_20 mFMO_24 mFMO_28
N290D R292 R292 R292 R292 R292
M353K E357 E357
K358R D374 D374 D374 D374 D374
L360E 2222 R356/R292
A365D K401 K401 K401
T370D 22222N394K N394K
N378D K300 K300 K300 K300 K300 K300 K300
P391D N394K
N394K T370D/D374 T370D/D374/P391D
L398K E366 E366 E366 E366 E366 2
To al
b
234457 8
a
Emp y cells indica e he absence o he mu a ed esidue lis ed o he le . Residues (na i e o mu an ) o ming a p edic ed sal b idge wi h he mu an esidue (fi s column)
a e lis ed, and i a mu an esidue does no o m a sal b idge in an mFMO a ian , i is ma ked by a minus sign (2).
b
The o al numbe s o mu an esidues in ol ed in o ming sal b idges a e shown.
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FIG 2 P edic ed in e helical sal b idges in ol ing o induced by mu a ed esidues in PROSS mu an models. (A) The s uc u e o
he na i e mFMO dime (PDBID 2XVH), which was used o gene a e he PROSS mu an models, is shown o he le . Chain A is
shown as a ibbon model, and chain B is shown in su ace iew. The la ge domain is shown in g ay, he small domain in whea , and he
co ac o s FAD and NADP
1
a e shown in yellow and g een s icks, espec i ely. The figu e o he igh shows he na i e mFMO chain A
o a ed 260° a ound he yaxis o he igh o emphasize he egion o he s uc u e whe e in e helical sal b idges a e in oduced in he
PROSS mu an s (do ed squa e). The emphasized egion ( esidues 274 o 407) includes be a s ands
b
17 o
b
20 and alpha helices
a
5 o
a
8. (B) The p edic ed new in e helical sal b idges o he 7 PROSS mu an models a e shown using he egion and o ien a ion desc ibed
in he legend o panel A. The wo models mFMO_14 and mFMO_15 ha e iden ical in e helical sal b idges and a e ep esen ed by one
s uc u al model. Residues o ming sal b idges a e shown as s icks, hyd ogen bonds be ween in e ac ing esidues a e shown as eal-
colo eddashes,andoxygenandni ogena omsa ecolo ed edandblue, espec i ely.Na i eandmu a ed esiduesa ecolo edblue
and o ange, espec i ely. Sal b idge esidues ha a e no p esen in na i e mFMO o he p e ious a ian a e labeled. The N378D-K300-
D351 sal b idge, which is p esen in all models, connec s helices
a
6and
a
7 ia he
b
18-
b
19 loop. The L360E-R292 sal b idge, p esen
in mFMO_28, is no in e helical bu connec s helix
a
6 o he
b
17-
b
18 loop. All sal b idges ha we e in oduced by PROSS a e p esen
in he consecu i e models, excep o L398K-E366, in oduced in mFMO_11, which is no p esen in mFMO_28.
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which had a T
50
o 40.6°C (Table 2). The T
50
alues inc eased wi h he numbe o mu a-
ions in he same manne as he T
m
, wi h a mode a e decline eco ded o mFMO_24 and
mFMO_28.
As p oduc ion o p o ein hyd olysa es is o en pe o med be ween pH 6.0 and 7.0 (14, 15),
we also assessed whe he enginee ing al e ed he pH op imum, which was p e iously de e -
mined obe8.5 o na i emFMO(6).Asseenby he esul sinTable2,allmFMO a ian shad
pH op ima be ween 7.5 and 8.0, sligh ly lowe han ha o na i e mFMO.
Al hough mFMO_20 did no ha e he lowes pH op imum among he mu an a ian s,
i displayed he g ea es imp o emen in empe a u e s abili y, as eflec ed by bo h T
m
and
T
50
. As mFMO_20 hus eme ged as he mos p omising a ian o indus ial applica ion,
we de e mined i s op imal empe a u e (T
op
) o enzyma ic ac i i y and compa ed i o ha
o na i e mFMO. To de e mine he T
op
, we assessed he specific ac i i y agains TMA a em-
pe a u es be ween 22°C and 50°C and pH 8.0 (Fig. S3). The T
op
o bo h na i e mFMO and
mFMO_20 was ound o be 40°C (Table 2). The T
op
o na i e mFMO was p e iously
epo ed o be 45°C (a pH 7.5) (6), bu he obse ed di e ences in he ac i i ies meas-
u ed a 40 and 45°C in bo h s udies we e ma ginal.
Enginee ing enzymes o inc ease s abili y o en comes wi h a ade-o o diminished ca a-
ly ic ac i i y (22). We he e o e pe o med a s eady-s a e kine ic analysis o na i e mFMO and
mFMO_20, using TMA as he subs a e wi h a fixed concen a ion o NADPH (Table 2). Unde
he condi ions es ed, he KTMA
mo na i e mFMO was 1.07
m
M and he kTMA
ca was 1.28 s
21
.
The KTMA
m alue o mFMO_20 was 0.83
m
M and he kTMA
ca alue was 0.93 s
21
, bo h sligh ly
lowe han hose o na i e mFMO. In e es ingly, he ca aly ic e ficiency (k
ca
/K
m
) o mFMO_20
emained almos iden ical o ha o na i e mFMO (Table 2).
mFMO_20 educes he TMA le el in salmon p o ein hyd olysa e by 95% a 65°C.
Since mFMO_20 demons a ed he mos p ominen inc ease in he mal s abili y, we
compa ed i s abili y o con e TMA o TMAO in a salmon p o ein hyd olysa e o ha o
na i e mFMO. The co ac o NADPH was supplemen ed, as he hyd olysa e did no con ain
su ficien amoun s o d i e he enzyma ic eac ion (6). Hea -calib a ed enzymes and 0.5 mM
NADPH we e added o salmon p o ein hyd olysa es (pH 6.1) and incuba ed o 1 h a 30°C,
50°C, and 65°C, ollowed by measu emen s o TMA and TMAO concen a ions (Fig. 3). When
ea ed wi h he na i e enzyme, he TMA le el in he hyd olysa e was educed by 52% a
30°C and 46% a 50°C, and only 29% educ ion was obse ed a 65°C. The mu an a ian
mFMO_20 ou pe o med na i e mFMO a all empe a u es, wi h a s iking 95% educ ion o
TMA a bo h 50°C and 65°C.
Ne wo k o no el pola in e ac ions s abilizes mFMO_20. To unde s and he s uc-
u al basis o he inc eased he mos abili y o mFMO_20, we c ys allized i wi h he
co ac o s FAD and NADPH. The c ys als we e indexed in he C222
1
spaceg oupandcon ained
he biological dime wi hin he asymme ic uni , wi h one FAD and one NADP
1
molecule
TABLE 2 Biochemical pa ame e s o mFMO and mu an a ian s
Enzyme
Mean alue ±SD o as indica ed
T
m
±95% CI (°C)
a
T
50
(°C)
b
T
op
(°C)
c
Op imal pH
d
K
m
(mM)
e
k
ca
(s
21
)
e
k
ca
/K
m
(mM
21
s
21
)
e
mFMO 46.2 60.2 40.6 60.4 40 8.5 1.07 60.13 1.28 60.03 1.20 60.12
mFMO_8 51.2 60.1 45.1 60.9 7.5
mFMO_11 51.7 60.1 47.1 61.2 7.5
mFMO_14 53.9 60.2 49.0 61.5 8.0
mFMO_15 54.5 60.1 49.6 60.4 7.5
mFMO_20 55.2 60.1 50.0 60.4 40 8.0 0.83 60.02 0.93 60.05 1.11 60.03
mFMO_24 52.0 60.1 49.2 60.2 7.5
mFMO_28 50.9 60.1 48.9 60.5 8.0
a
Mel ing cu es we e ob ained by CD a pH 7.5, and mel ing empe a u e was es ima ed by ou -pa ame e logis ic eg ession o he mel ing cu e.
b
The empe a u e whe e hal o he enzyme ac i i y was los (T
50
) was measu ed a pH 7.5, and he epo ed da a a e he mean alues 6s anda d de ia ions (SD) om h ee
independen expe imen s.
c
Op imal empe a u e (T
op
) was de e mined a pH 8.0 o mFMO and mFMO_20.
d
Op imal pH o TMA con e sion was de e mined a 22°C.
e
S eady-s a e kine ic measu emen s we e pe o med wi h a ious concen a ions o TMA (Sigma-Ald ich) as he subs a e and a fixed NADPH concen a ion (200
m
M) a
23°C, pH 8.0. Repo ed da a a e he mean alues 6SD om wo biological eplica es.
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bound pe ca aly ic si e. The c ys al s uc u e o he mFMO_20/FAD/NADP
1
complex was
sol ed a 1.62-Å esolu ion, e ealing a s uc u e highly simila o ha o na i e mFMO (PDB
ID 2XVH)(19), as eflec ed by a calcula ed oo mean squa e de ia ion (RMSD) o 0.27 Å (on
445 C
a
a oms). The small domain con ains 4 mu a ions, and he la ge domain con ains he
o he 16 (Fig. 4). Compa ed o na i e mFMO, mFMO_20 has a ne cha ge change o 24.
In e es ingly, hal o he mFMO_20 mu a ions a e loca ed in a 46-amino-acid subsequence
FIG 3 Enzyma ic TMA con e sion in salmon p o ein hyd olysa e. Salmon p o ein hyd olysa es (pH 6.1)
we e incuba ed o 1 h a 30, 50, and 65°C wi h no enzyme (con ol), mFMO, o mFMO_20, all supplemen ed
wi h 0.5 mM NADPH. TMA and TMAO le els we e de e mined using UHPLC wi h he EVOQ Eli e iple
quad upole mass spec ome e . The expe imen was pe o med wi h h ee biological eplica es, and he
plo shows he mean TMA (g ay) and TMAO (yellow) le els (ppm) in s acks wi h s anda d de ia ions (SD)
ep esen ed by e o ba s.
FIG 4 C ys al s uc u e o mFMO_20. The s uc u e o he mFMO_20 monome (PDB ID 8B2D) (chain
A) is shown as a ibbon model, wi h he la ge domain colo ed in g ay and he small domain colo ed
in whea . The co ac o s FAD and NADP
1
a e shown as yellow and g een s icks, espec i ely. The side
chains o he 20 mu a ed esidues a e ep esen ed as o ange s icks, and oxygen and ni ogen a oms
a e colo ed ed and blue, espec i ely. The alpha ca bons o he in oduced glycine esidues a e
shown as sphe es. The 5 esidues ha a e new compa ed o he sequence o mFMO_15 a e labeled
in g een. The h ee
a
-helices whe e he 10 o 20 mu a ed esidues a e loca ed a e labeled in ed.
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(M353Q o L398K) o a egion in he la ge domain con aining h ee helices:
a
6 (K345 o T361),
a
7 (A365D o M382), and
a
8 (I390 o N406) (Fig. 3). S uc u al analysis e ealed ha fi e o
hese mu a ed esidues o m new sal b idges in ol ing six na i e esidues (K358R-D374,
L360E-R356, A365D-K401, N378D-K300-D351, and L398K-E366), ou o which o m in e helical
in e ac ions(Fig.5A,Table3), husconfi ming he PROSS model p edic ions (Fig. 2). Helices
a
6
and
a
7 a e di ec ly connec ed by K358R-D374 and indi ec ly connec ed, ia he
b
18-
b
19
loop, by N378D-K300-D351. Helices
a
7and
a
8 a e connec ed by A365D-K401 and L398K-
E366. Two new hyd ogen bonds in ol ing side chains a e also in oduced, one o ming an
in ahelical bond (
a
6; M353Q-R356) and he o he an in e helical bond (
a
7-
a
8; T370D-N394)
(Table 3). In addi ion o he sal b idges di ec ly in oduced by mu a ed esidues, mFMO_20
has 4 sal b idges in ol ing na i e-only esidues ha a e no p esen in he na i e mFMO
s uc u e (PDB ID 2XVH) ha we used as s a ing poin (Fig. 5A). Howe e , h ee o hese sal
FIG 5 S abilizing sal b idges in na i e mFMO and mu an mFMO_20. (A) Chain A o he mFMO_20 s uc u e is shown as
a ibbon model, wi h he small and la ge domains colo ed in whea and g ay, espec i ely, and he co ac o s FAD and NADP
1
shown as yellow and g een s icks, espec i ely. Residues o ming sal b idges ha a e unique o mFMO_20 a e shown as s icks,
hyd ogen bonds be ween in e ac ing esidues a e shown as eal-colo ed dashes, and oxygen and ni ogen a oms a e colo ed ed
and blue, espec i ely. Na i e and mu a ed esidues a e colo ed blue and o ange, espec i ely. The esidues o sal b idges
in ol ing mu an esidues a e labeled. The h ee alpha helices whe e he mu a ed esidues a e loca ed a e labeled in ed. (B)
Chain A o he mFMO s uc u e (PDB ID 2XVH) is isualized essen ially as desc ibed in he legend o panel A. Residues ha o m
sal b idge pai s in bo h he na i e mFMO and mu an mFMO_20 a e colo ed sla e blue, and he lone pai which is unique o
mFMO is colo ed cyan. (C) The biological dime o na i e mFMO is shown in B- ac o pu y ep esen a ion. Red colo s and la ge
diame e so he ubesindica eflexible egions wi h highe B- ac o s, in con as o blue colo s wi h small diame e s, indica ing
well-o de ed egions wi h lowe B- ac o s. The loca ion o he loop be ween
b
-s ands 11 and 12 is indica ed. Chain B is colo ed
whi e. (D) The biological dime o mu an mFMO_20 is isualized as desc ibed in he legend o panel C. The iew o all s uc u es
has been o a ed 260° a ound he yaxis compa ed o he iew in Fig. 4.
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b idges a e p esen in a leas 4 o he o he 11 mFMO PDB s uc u es. The p esence o he
appa en ly unique R19-E26 sal b idge in mFMO_20 may eflec di e ences in c ys alliza ion
condi ions. All bu one o he sal b idges iden ified in na i e mFMO a e also p esen in
mFMO_20 (Fig. 5B). The B- ac o p ofile o mFMO_20 is di e en om ha o na i e mFMO
(Fig. 5C and 5D). The lowe B- ac o s obse ed nea he en ance o he ac i e si e in ou
c ys allog aphic analysis a e consis en wi h he inc eased he mal and kine ic s abili y o
his design. The egion wi h he highes B- ac o in mFMO_20 is he loop be ween
b
-s ands
11 and 12, which is loca ed a he en ance o he ac i e si e. In con as , se e al loops in he
na i e mFMO display highe B- ac o s han he
b
11-
b
12 loop.
DISCUSSION
We ha e p e iously shown ha mFMO can con e he malodo ous TMA molecule in o
he odo less TMAO in a salmon p o ein hyd olysa e (6). To make mFMO mo e sui able o
such indus ial applica ions, which ypically ake place a empe a u es anging om 45°C o
60°C (14, 15), we employed he PROSS algo i hm o imp o e i s he mal s abili y. All 7 mu an
a ian s o mFMO we e unc ional enzymes wi h inc eased he mos abili y (Table 2). The bes
a ian was mFMO_20, which displayed he highes inc ease in empe a u e s abili y (Table
2). The c ys al s uc u e o mFMO_20 demons a ed ha he o e all s uc u e o his mu an
a ian washighlysimila o ha o na i emFMO,bu i also e ealednews uc u al ea u es
ha could explain he inc eased s uc u al s abili y. The mos s iking new ea u es we e fi e
sal b idges in ol ing mu a ed esidues, ou o which o med s abilizing in e helical connec -
ing b idges (Fig. 5A). Al hough PROSS ailed o co ec ly model he in ahelical sal b idge
be ween L360E and R356 and p edic ed an in ahelical sal b idge be ween N290D and R292
ha was no obse ed in he c ys al s uc u e, all ou in e helical sal b idges we e co ec ly
modeled (Fig. 2), hus emphasizing he quali y o he PROSS p edic ions. The only in e helical
sal b idge ha was p esen in all 7 PROSS models was he N378D-K300-D351 b idge, which
connec s
a
6 o
a
7 ia he
b
18/
b
19 loop (Fig. 2). The N378D mu a ion leads o he eplace-
men o a hyd ogen bond be ween N378 and K300 in he na i e mFMO s uc u e by a s on-
ge sal b idge be ween N378D and K300 and also induces K300 o o m a sal b idge wi h
D351, which is no p esen in he na i e s uc u e. The ac s ha he la ges inc ease in em-
pe a u e s abili y om one a ian o he nex was obse ed going om he na i e mFMO o
mFMO_8 and ha mFMO_8 only con ains one in e helical sal b idge may sugges ha
N378D is a key s abilizing mu a ion. Howe e , i is di ficul o decon olu e he e ec o he
in oduced in e helical sal b idges om he e ec s o he o he mu a ions. Ne e heless, he
obse ed g adual inc ease in empe a u e s abili y om mFMO_8 o mFMO_20 coincides
wi h a g adual inc ease in he numbe o such b idges om 1 in mFMO_8 o 4 in mFMO_20
(Fig. 2), sugges ing ha hese elec os a ic in e ac ions ha e an impo an ole in inc easing
empe a u e s abili y. These esul s a e in line wi h he ecen ly published communi y-wide
PROSS e alua ion, whe e a co ela ion be ween he numbe o mu a ions and gain o he mal
s abili y was obse ed (21). Howe e , despi e in oducing 4 and 8 mo e mu a ions, leading o
TABLE 3 New pola in e ac ions di ec ly in ol ing o induced by side chain a oms o
mu a ed esidues in mFMO_20
Hyd ogen dono
a
Hyd ogen accep o
a
Dis ance (Å)
b
S uc u al loca ion
c
Dono Accep o
K300 (N
z
) D351 (O
d
2
) 3.0 Loop
b
18-
b
19
a
6
K300 (N
z
) N378D (O
d
2
) 2.8 Loop
b
18-
b
19
a
7
R356 (N
«
/N
h
2
) M353Q (O
«
1
) 2.8/2.7
a
6
a
6
R356 (N
h
1
) L360E (O
«
2
) 3.4
a
6
a
6
K358R (N
h
1
/N
h
2
) D374 (O
d
2
) 3.0/2.8
a
6
a
7
N394 (N
d
2
) T370D (O
d
1
) 3.4
a
8
a
7
L398K (N
z
) E366 (O
«
2
) 3.1
a
8
a
7
K401 (N
z
) A365D (O
d
2
) 2.7
a
8
a
7
a
Hyd ogen dono and accep o a oms o he amino acid side chains a e indica ed in pa en heses.
b
The dis ances be ween hyd ogen dono and accep o a oms o he amino acid side chains a e shown.
c
The s uc u al loca ion is indica ed by he seconda y s uc u e elemen .
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wo mo e sal b idges p edic ed o connec seconda y s uc u e elemen s in mFMO_24 (bo h
be ween
a
7and
a
8) and mFMO_28 (one be ween
a
7and
a
8 and one be ween
a
6and he
b
17/
b
18 loop), he empe a u e s abili y o hese a ian s dec eased sligh ly compa ed o he
empe a u e s abili y o mFMO_20. One possible explana ion is ha he in ica e ne wo ks o
sal b idges in mFMO_24 and mFMO_28, which also in ol e di ec b idges be ween mu a ed
esidues, a e inco ec ly p edic ed by PROSS.
In a p e ious e o o iden i y mu a ions ha con e inc eased he mos abili y o mFMO,
Lon
ca and colleagues used he FRESCO p o ocol o p edic s abilizing single mu a ions (16).
The FRESCO analysis yielded 140 single-mu an candida es ha we e exp essed, pu ified,
and sc eened o inc eased he mos abili y, and 14 o hese displayed an appa en inc ease
in mel ing empe a u e o .1°C. The wo mu a ions M15L and S23A we e combined, and
he esul ing mFMO a ian had a 3°C inc ease in mel ing empe a u e. Adding addi ional
s abilizing single mu a ions did no u he inc ease he mos abili y. In line wi h wha we
obse ed wi h mFMO_20, no majo e ec s we e obse ed on he kine ic pa ame e s o he
mFMO M15L/S23A a ian . FRESCO has also been used o s abilize he Rhodococcus sp.
s ain HI-31 cyclohexanone monooxygenase (23), which also belongs o he FMO amily. In
his case, hal o he 128 sc eened single-mu an a ian s had modes s abilizing e ec s.
These we e combined, using a shu fled lib a y design s a egy, in o a a ian ca ying 8
mu a ions (M8B), which inc eased he un olding empe a u e by 13°C. In con as o he
PROSS mFMO mu an a ian s, he FRESCO-de i ed mu a ions did no appea o o m new
sal b idges in ei he M8B o mFMO M15L/S23A.
We enginee ed mFMO o make i mo e sui able o indus ial applica ions like emo ing
TMA in salmon p o ein hyd olysa es. The mFMO_20 a ian was selec ed as he bes candi-
da e due o i being he mos he mos able a ian o he se en designs. In addi ion, he
op imal pH o mFMO_20, pH 8.0, was sligh ly lowe han ha o he na i e mFMO, pH 8.5,
which may also con e an ad an age in indus ial applica ions (e.g., he pH o he salmon
p o ein hyd olysa e was 6.1). In ac , he op imal pH o all 7 mu an a ian s was be ween
7.5 and 8.0 (Table 2), bu wi h no disce nible co ela ion wi h changes in cha ge o pI. The
op imal empe a u e o mFMO_20 did no inc ease compa ed o ha o na i e mFMO. S ill,
his mino disad an age o mFMO_20 was clea ly ou weighed by he beneficial p ope ies
o inc eased s abili y when i was es ed o i s abili y o con e TMA o TMAO in he salmon
p o ein hyd olysa e (Fig. 3). A bo h 50°C and 65°C, mFMO_20 was supe io o na i e mFMO
in emo ing TMA, elimina ing 95% o TMA, and i also appea ed o pe o m bes a 30°C.
These esul s demons a e ha mFMO_20 is indeed mo e sui able o indus ial applica ions
han he na i e mFMO. Howe e , he e a e s ill impo an hu dles ha mus be o e come
be o e his Tmm enzyme can be inco po a ed in o an indus ial p ocess, especially i s de-
pendence on he uns able and expensi e co ac o NADPH. The ac ha we and o he s
ha e demons a ed ha he Tmms can be enginee ed opens he possibili y o co ac o en-
ginee ing, which can be used o make he enzyme accep mo e cos -e ficien co ac o s. An
al e na i e o complemen a y s a egy is o b ing down cos by egene a ing he co ac o ,
e.g., by using glucose dehyd ogenase (24).
The cu en wo k has demons a ed ha he PROSS me hod could success ully p edic
mFMO a ian s wi h inc eased he mos abili y. We did no obse e any appa en imp o e-
men in exp ession o solubili y le els, which equen ly ollow imp o emen s in s abili y,
which may be due o he ac ha he na i e mFMO enzyme is al eady eadily exp essed
in soluble o m. All 7 a ian s p oposed by PROSS showed inc eased he mos abili y, wi h
p ope ies compa able o hose o enginee ed FMO enzymes ob ained a e sc eening
mo e han 100 single-mu an a ian s ollowed by lib a y shu fling o a ional enginee ing
(16, 23). The mFMO_20 a ian wi h i s imp o ed s abili y may be applicable o indus ial
use as is, because i can educe he majo i y o TMA p esen in fish hyd olysa es. I can
also se e as an excellen s a ing poin o a ional enginee ing o u he imp o e i s ca a-
ly ic e ficiency o o co ac o enginee ing o make i accep mo e cos -e ficien co ac o s.
MATERIALS AND METHODS
P o ein s abiliza ion mu agenesis using he PROSS Web se e . The P o ein Repai One-S op
Shop (PROSS) se e (h ps://p oss.weizmann.ac.il/) was used o p edic a ian s o mFMO wi h inc eased s abili y
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