of 43
ACS Synthetic Biology
Supporting information for
A general tool for engineering the
NAD/NADP cofactor preference of
oxidoreductases
JKB Cahn, CA Werlang, A Baumschlager, S
Brinkmann
-
Chen, SL Mayo, and FH Arnold
Supporting
Table 1.
NADP
-
to
-
NAD cofa
ctor specificity reversal. Structures followed by (h) are homology models, while those followed by another PDB accession
code use the cofactor from that protein and (m) denotes a structure of a mutant protein. Mutations indicated in italics are d
istal to t
he 2' position. For citation
information, see
Supporting
Material 2
.
Study
Protein
Structure
Best
Variant
Final
Specificity
a
Specificity
Change
b
Log Relative
Activity
c
(Banta et al. 2002)
C. glutamicum
DKR
1A80
R238H
?
?
?
(Baroni et al. 2012)
P. falcipa
rum
FDNR
2OK7
Y258F
0.67
4.7x10
1
-
1.2
(Bastian et al. 2011)
E. coli
KARI
3ULK
A71S, R76D, S78D,
Q110V
1
90
5.4x10
4
-
0.07
(Brinkmann
-
Chen et al. 2013)
S.
sp.
KARI
3ULK
(h)
A71S, R76D, S78D,
Q110V
64
1.1
x10
5
-
2.1
(Brinkmann
-
Chen et al. 2013)
S. exigua
KARI
4KQW
S61D, S63D,
I95V
88
7.8x10
3
-
1.6
(Brinkmann
-
Chen et al. 2013)
M. aeolicus
KARI
4KQW
(h)
G50D, S52D
120
1.2x10
3
-
0.84
(Brinkmann
-
Chen et al. 2013)
L. lactis
KARI
4TSK
(h)
V48L, R49P, K52L, S53D,
E59K
,
T182S
,
E320K
15
0
2.3x10
4
0.0
4
(Brinkmann
-
Chen e
t al. 2013)
A. acidocaldarius
KARI
4TSK
R48P, S51L, S52D,
R84A
110
4.5x10
2
-
1.5
(Chen et al. 1995)
E. coli
IDH
4AJ3
C201I
,
C332Y
, K344D, Y345I, V351A,
Y391K, R395S
20
0
1.4x10
6
-
1.5
(Dambe et al. 2006)
S. morelense
AFDH
2GLX
A13G
, S33D
1
4
?
-
0.79
(Döhr e
t al. 2001)
H. sapiens
P450R
3QFS
W676A
0.2
4
1.0x10
3
-
0.55
(Elmore and Porter 2002)
R. norvegicus
P450R
1AMO
W677A
1.
2
5.3x10
4
-
1.8
(Eppink et al. 1999)
P. fluorescens
PHBH
1K0J
(m)
R33S
,
Q34R
,
P36R
,
D37A
, Y38E
3.1
5.0x10
4
-
2.0
(Fasan et al. 2011)
B. me
gaterium
P450R
4DQL
R966N, K972H, Y974F, W1046D
0.6
2
4.4x10
2
-
1.
1
(Gand et al. 2016)
S.
sp. IRED
3ZHB (h)
S37V, K40A
5.5
1.8x10
2
-
1.6
(Kamerbeek et al. 2004)
P. fluorescens
HAPMO
2YLR
(h)
K439F
0.6
2
4.3x10
2
-
2.0
(Kamerbeek et al. 2004)
A.
sp.
CHMO
4RG3
K326A
0.
10
5.5x10
1
-
2.9
(Katzberg et al. 2010)
S. cerevisae
DKR
4PVD
N9E
0.86
1.2x10
2
-
2.9
(Khoury et al. 2009)
C. boidinii
XR
1K8C
(h)
K272G, S273G, N274D
>1
?
-
1.1
(Kristan et al. 2007)
C. lunatus
BHSDH
3QWF
Y49D
7.
8
?
-
4.
2
(Liang et al. 2007)
P. sti
pitis
XR
1K8C
(h)
K270R, N272D
2.9
9.3
-
1.
2
(Maddock et al. 2015)
E. coli
CaADH
1KEV
(h)
G198D, S199V, P201E, Y218A
>1
?
-
4.
6
(Maurer et al. 2005)
B. megaterium
P450R
4DQL
R966D,
W1046S
0.37
2.3
x10
2
-
0.40
Supporting
Table 1
.
continued
Study
Protein
Str
ucture
Best Mutant
Final Specificity
a
Specificity
Change
b
Log Relative
Activity
c
(Medina et al. 2001)
A.
PCC7119
FDNR
2BSA
S223D
0.12
8.1x10
3
-
5.
4
(Nakanishi et al. 1997)
M. musculus
CR
1CYD
T38D
3
1
1.3x10
3
-
0.51
(Paladini et al. 2009)
P. sativum
FDNR
4
AF7 (1QGA)
Y308S
0.0
2
3.3x10
2
-
1.6
(B. Petschacher et al. 2005)
C. tenuis
XR
1K8C
K274R, N276D
1.2
1.9x10
1
-
1.
2
(Pick et al. 2014)
E. coli
AdhZ3
1YQD
(h)
S199N, S200N, N201D
1.2
6.9x10
1
-
0.75
(Pick et al. 2014)
E. coli
AdhZ2
1UUF
(1YQD)
T205D, T206I, S2
07N
2.
2
3.8x10
1
0.00
(Rane and Calvo 1997)
E. coli
KARI
3ULK
R68D, K69L, K75V, R76D
31
5.8x10
4
-
0.5
5
(Rodriguez
-
Arnedo et al. 2005)
H. volcanii
IDH
1AI2
(h)
R291S, K343D, Y344I, V350A,
Y390P
>1
?
-
0.7
7
(Rosell et al. 2003)
R. perezi
ADH8
1P0F
G223D, T22
4I, H225N
7
600
8.1x10
4
0.55
(Schepens et al. 2000)
S. bicolor
MDH
7MDH (1CIV)
G84D, S85I, R87Q, S88A
12
2.1x10
4
-
0.96
(Scrutton et al. 1990)
E. coli
GTR
1GET
A179G
,
A183G
, V197E, R198M,
K199F, H200D, R204P
8.1
1.8x10
4
-
1.
5
(Shiraishi et al. 1998)
N. cra
ssa
CbR
S920D, R932S
65
7.2x10
4
-
2.6
(Takase et al. 2014)
S.
sp.
A1
-
R
3AFN
H37N, G38S, R39H, K40V, A41D
>1
?
-
3.2
(Yaoi et al. 1996)
T. thermophilus
IDH
2D1C
R231A, K283D, Y284I, N287G,
V288I
, I290A
6
8
5.2
x10
4
-
1.1
(Zeng et al. 2009)
P. stipitis
XR
1K8
C
(h)
K21A, N272D
>1
?
-
0.
10
(L. Zhang et al. 1999)
V. harveyi
ALDH
1EZ0
T175E
1
30
4.8x10
3
-
0.76
(R. Z. Zhang et al. 2009)
C. parapsilosis
SCR
3CTM
(1CYD)
S67D, P69D
0.31
4.6
-
0.18
This study
A. thaliana
GR
3DOJ (3PEF)
R31L, T32K, K35D, C68R
2.4
3.3x10
1
-
0.71
This study
S. cerevisae
CinADH
1PIW
S210D, R211P, K215E, S253P
2.0
6.6x10
1
0.77
This study
T. emersonii
XR
1K8C (h)
S272G, N273G, R277Y, Q280E
5.2
4.8x10
3
-
1.5
This study
T. maritima
FeADH
1VHD
G36E, S38N, S39G
43
8.4x10
1
0.47
Catalytic efficien
cy,
CE
, is given as
k
cat
/
K
M
when available, or
v
max
/
K
M
otherwise.
a
Final specificity is defined as
푪푬
풎풖풕
푵푨푫
푪푬
풎풖풕
푵푨푫푷
b
Specificity change is defined as
푪푬
풎풖풕
푵푨푫
푪푬
풎풖풕
푵푨푫푷
푪푬
푾푻
푵푨푫
푪푬
푾푻
푵푨푫푷
c
Log relative activity is defined as
퐥퐨퐠
(
푪푬
풎풖풕
푵푨푫
푪푬
푾푻
푵푨푫푷
)
Supporting
Table 2
.
NAD
-
to
-
NADP cofactor specificity reversal. Structures followed by (h) are homology models, while those followed by another PDB
accession code use the cofactor from that protein and (m) denotes a structure of a mutant protein. Mutati
ons indicated in italics are distal to the 2' position. For
citation information, see
Supporting
Material 2
.
Study
Protein
Structure
Best Mutant
Final
Specificity
a
Specificity
Change
b
Log Relative
Activity
c
(Ashida et al. 2004)
S.
sp
AlaDH
2VHW
(h)
D198A
1
4
3.4x10
4
-
0.96
(Bernard et al. 1995)
L
.
delbruckii
LDH
1J49
D175A
1.0
4.4x10
1
-
0.96
(Bocanegra et al. 1993)
E
.
coli
DHLDH
4JQ9 (1GEU)
E205V, M206R, F207K,
D208H
,
P212R
>1
?
0.6
7
(Bubner et al. 2008)
P
.
fluorescens
M2DH
1M2W
E68K, D69A
1
9
4.5x10
3
0.7
7
(Capone et al. 2011)
C
.
symbiosum
GDH
1BGV (4XGI)
F238S, P262S
0.32
5.8x10
1
-
3.3
(Chen et al. 1996)
T
.
thermophilus
IMDH
2ZTW
N/A
1000
8.7x10
4
0.1
9
(Clermont et al. 1993)
B
.
stearothermophilus
GAPDH
3CMC
D32A, L187A, P188S
1.6
?
-
1.
7
(Cui et al. 2015)
G
.
oxydans
Gox2181
3AWD
(2WDZ)
Q20R, D43S
1.5
?
0.07
(Ehrensberger et al. 2006)
G
.
oxydans
XDH
1ZEM
D38S, M39R
>1
?
0.
70
(Ehsani et al. 2009)
S
.
cerevisae
BDH
2D8A
(h)
E221S, I222R, A223S
>1
?
0.0
6
(Feeney et al. 1990)
B
.
stearothermophilus
LDH
1LDN
D53S
0.15
3.4
-
1.3
(Friesen et al. 1996)
P
.
mevalonii
HMG
-
CoAR
4I4B
D146A, L148K
0.1
4
7.6x10
4
-
3.
7
(Galkin et al. 1997)
T
.
intermedius
LuDH
1LEH
(h,1BW9)
D203A, I204R,
D210R
74
?
-
1.6
(Gul
-
Karaguler et al. 2001)
C
.
methylica
FDH
2FSS (2NAD)
D195S
0.02
6.1x1
0
3
-
1.
5
(Hoelsch et al. 2013)
M
.
vaccae
FDH
2GSD
(h)
C145S
, A198G, D221Q,
C225V
13
?
?
(Holmberg et al. 1999)
B
.
stearothermophilus
LDH
1LDN
I51K, D52S
2.2
4.9x10
1
-
1.
6
(Hong et al.
2016)
B.
cereus
AlDH
4PT0
E194S
0.47
2.1x10
1
-
0.46
(Hsieh et al. 2006)
H
.
sapiens
m
-
NAD
-
ME
1PJ3
Q362K
3.2
2.9x10
2
-
0.53
Supporting
Table 2
.
continued
Study
Protein
Structure
Best Mutant
Final
Specificity
a
Specificity
Change
b
Log Relative
Activity
c
(Jensen et al. 2013)
S
.
maltphilia
SMFMO
4A9W
(2XLP)
H194T
0.9
7
1.5
-
0.2
2
(Ma et al. 2010)
K
.
pneumonia
PDOR
3OX4
(h)
D41G
1.5
6
?
-
0.6
6
(Marohnic et al. 2003)
R
.
norvegicus
CB5R
1IB0
D239T
10
4.1x10
4
-
0.6
7
(Miller et al. 2006)
E
.
coli
IMDH
1CM7
(2ZTW)
K100R
,
A229T
,
D236R
,
L248M
,
D289K, I290Y, A296V,
G337Y
370
5.1x10
4
-
0.2
3
(
Nishiyama et al. 1993)
T
.
flavus
MDH
1BMD
E41G, I42S, P43E, Q44R, A45S,
M46F
,
K47Q
24
5.2x10
2
-
0.47
(Petschacher
et al. 2014)
S
.
mutans
NOX
2BC0
(h,2CDU)
D192A, V193R, V194H, A199R
10
6.4x10
4
0.5
6
(Serov et al. 2002)
S
.
cerevisae
FDH
2NAD
(h)
D196A, Y197
R
2.3
?
-
3.
8
(Takase et al. 2014)
S.
sp.
A1
-
R’
4TKM
T16S, E17Q, N37H, S38G, H39R,
V40K, D41A
8
5
1.1x10
3
0.6
7
(Watanabe et al. 2005)
P
.
stipitis
XDH
1PL6
(h)
D207A, I208R, F209T
2.6
1.1x10
4
0.0
5
(Woodyer et al. 2003)
P
.
stutzeri
PDH
4E5K
E175A, A176R
2.
8
3.0x10
2
1.0
(Zheng et al. 2013)
B
.
subtilis
InDH
3NT2
A12K
, D35S, V36R
4.
8
?
0.03
Catalytic efficiency,
CE
, is given as
k
cat
/
K
M
when available, or
v
max
/
K
M
otherwise.
a
Final specificity is defined as
푪푬
풎풖풕
푵푨푫
푪푬
풎풖풕
푵푨푫푷
b
Specificity change is defined as
푪푬
풎풖풕
푵푨푫
푪푬
풎풖풕
푵푨푫푷
푪푬
푾푻
푵푨푫
푪푬
푾푻
푵푨푫푷
c
Log relative activity is defined as
퐥퐨퐠
(
푪푬
풎풖풕
푵푨푫
푪푬
푾푻
푵푨푫푷
)
Supporting
Figure 1
.
Average number of residues of each amino acid comprising the specificity
-
determining pockets for NAD
-
bound and NADP
-
bound
structures in the PDB, compiled from a representative set of 499 NAD
-
bound structures and 463 NADP
-
bound
structures selected on the basis of sequence
identity and resolution. The criteria for selecting ‘specificity
-
determining’ residues are laid out in
Supporting
Material 1
. It is important to note that NADP
binding pockets on average comprise more residues,
3.58 vs 2.89 on average for NAD binding pockets.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Average number of CSR
-
SALAD determined
specificity
-
determining contacts per binding
site
NAD
NADP
Supporting
Figure 2
.
The same data as
Supporting
Fig. 1
, but broken out into the four most important structural classes (see
Supporting
Fig. 3
and
Supporting
Material 1
). For
clarity, only the five most utilized amino acids for each of the two cofactors are shown for each category.
0.0
0.1
0.1
0.2
Gly
Glu
Ser
Lys
Asn
Asp
Ala
Edge
NAD
NADP
0.0
0.2
0.4
0.6
Ile
Gln
Gly
His
Pro
Val
Ala
Arg
Tyr
Face
NAD
NADP
0.0
0.1
0.2
0.3
Gly
Glu
Ser
Lys
Asn
Thr
Arg
Bidentate
NAD
NADP
0.0
0.2
0.4
0.6
Gly
Glu
Ser
Lys
Asn
Val
Thr
Asp
Arg
Other
NAD
NADP
Supporting
Figure 3
.
Examples of the six structural classifications used by CSR
-
SALAD. For more details, see the
Supporting
Material 1
. Figures
are from
structures 1VC2, 1AMO, 1AMO, 1EZ0, 1CYD, and 1CYD.
Supporting
Figure 4
.
The web interface of CSR
-
SALAD (a) and an example of the corresponding output (b).