1
Supplemental Information
1:
S
pecificity loop length and KARI class of 3,422 unique
KARIs retrieved from Pfam. Four additional Class I KARIs had other specificity loop
lengths. Green indicates enzymes with representative structures published prior to this
study.
Loop Length
6
7
12
Class I
595
2260
5
Class II
0
20
538
2
Supplemental Information 2: Refinement Statistics for structures presented in this paper.
Values in parentheses represent statistics from the highest
-
resolution shell.
Aa_KARI
_holo
4TSK
Ua_KARI
_holo
4XDY
Ia_KARI
_holo
4XDZ
I
a_KARI_apo
4XEH
Av_KARI
_met
4XIY
Data Collection
Space Group
P
2 3
P
2
1
2
1
2
P
2
1
P
2
1
2
1
2
P
2
1
3
Cell Dimensions
a
,
b
,
c
(Å)
124.10, 124.10,
124.10
141.75, 148.60
,
49.70
54.58, 90.75,
69.53
67.48,
112.35
,
46.07
185.05, 185.05,
185.05
α
,
β
,
γ
(°)
90, 90, 90
90, 90, 90
90, 100.3, 90
90, 90, 90
90, 90, 90
Resolution (Å)
124.10
-
2.50 (2.57
-
2.50)
102.57
-
1.
5
4
(
1.58
-
1.54)
68.42
-
1.15 (1.21
-
1.15)
57.85
-
1.39 (1.43
-
1.39)
82.76
-
2.50 (2.64
-
2.50)
R
p.i.m
(%)
5.3 (61.8
)
4.4 (49.8
)
5.4 (64.3)
2.2 (107.9)
5.7 (31.3)
Mn(I)/sd
8.9 (0.8)
11.1 (1.5)
6.9 (0.9)
13.6 (0.6)
10.9 (2.6)
Completeness (%)
97.7 (89.1)
97.3 (71.8)
95.0 (82.4)
96.8 (76.0)
99.5 (99.9)
Redundancy
3.4 (2.9)
4.5 (4.5)
3.0 (2.5)
3.6 (3.9)
3.5
(3.6)
Refinement
No. reflections
22,066
145,320
224,341
65,375
72,509
R
work
/R
free
(%)
15.6/18.2
(21.2/27.2)
17.9/20.9
(
39.1/38.4)
15.8/19.0
(33.2/34.1)
18.0/23.5
(38.2/38.0)
19.4/24.4
(28.7/32.1)
No. atoms
Protein
2,585
5,296
5,139
2,561
10,017
Ligand/ion
61
126
178
0
48
Water
46
359
595
101
40
R
MSD
Bond lengths (Å)
0.018
0.023
0.024
0.019
0.015
Bond angles (°)
1.998
2.038
2.461
1.930
1.719
Ramachandran map
analysis
Favored
313
653
589
309
1,194
A
l
lowed
17
20
29
13
99
Outliers
1
0
0
0
23
3
Supplemental Information
3: Sequence alignment of
crystallized KARIs.
Helices are
colored in magenta, and
β
-
strands in cyan. Yellow indicates special features, labeled
with the following abbrevia
tions:
motif = diphosphate binding motif; specific. =
specificity loop; m = metal
-
binding residue; n = nicotinamide amide
-
binding residue; h =
hinge point (when unambiguously definable). Metal
-
binding and nicotinamide amide
-
binding residues are shown only for on
e active site in the
Class I proteins. The knotted
domain of the Class I KARIs has been duplicated for clarity; duplicate residues are
shown with grey text.
4
Ia_KARI
----------------------
AKIYK
DED
I
---
S
L
-
EPIK
N
-
KT
IAIL
GYGSQG
RAWALNLRDS
G
------
Ua_KARI
----------------------
MEILH
DED
V
---
DD
-
SILR
D
-
K
TIAVM
GYGAQG
DAQANCLKDS
G
------
Av_KARI
----------------------
MKVYY
DKD
C
---
D
L
-
SIIQS
-
KK
VAII
GYGSQG
HAHACNLKDS
G
------
Pa_KARI
----------------------
MRVFY
DKD
C
---
DL
-
SIIQG
-
KK
VAII
GYGSQG
HAHACNLKDS
G
------
Aa_KARI
-----------------------
K
IY
YDADI
---
S
I
-
QPLAD
-
K
RIAVI
GYGSQG
HAHAQNLRDS
G
------
Se_KARI
-----------------------
T
IL
YEQDV
---
D
P
-
KVIQG
-
LK
VGII
GYGSQG
HAHALNLMDS
G
------
Ec_KARI
---
MAN
YFNT
LN
LRQQLAQLG
KC
RF
M
GRDEF
-
A
DG
A
-
SYLQ
G
-
K
KVVIV
GCGAQG
LNQGLNMRDS
G
------
So_KARI
ATTFDFDSSVFKK
EKVTL
SG
HDEYIVR
GG
RNLFPLLPDAF
KGIK
QIGVI
GWGSQA
PAQAQNLKDSLTEAK
SD
Os_KARI
---
LDFDTSVFNKE
KVSL
AG
HEEYIVR
G
GRNLFPLLPEAF
KGIK
QIGVI
GWGSQG
PAQAQNLRDSLAEA
KSD
Features b1 motif aA
Ia_KARI L
NVVVG
LER
-----
QGDS
WRRA
IDDGF
-----
KPMY
TKDAVAI
AD
IIVF
LVP
DMVQKSLWLNSVKDFM
KKGA
Ua_KARI IN
VVIG
ETEILGGNKNPS
WEKAKED
GF
-----
E
VLP
IDKAAEK
GD
VVHI
LLP
DEVQPAIYENQIKP
QLKAGK
Av_KARI VD
VYVG
LRA
-----
GSAS
VAKAEAH
GL
-----
TVKS
VKDAVAA
AD
VVMI
LTP
DEFQGRLYKDEIEPN
LKKGA
Pa_KARI VD
VTVG
LRS
-----
GSAT
VAKAEAH
GL
-
----
KVA
DVKTAVAA
AD
VVMI
LT
PDEFQGRLYKEEIEPNL
KKGA
Aa_KARI F
DVVIG
LRP
-----
G
-
SS
WAKAEAD
GF
-----
R
VMA
VGDAVEES
D
VIMI
LL
PDERQPAVYEREIRPYL
TAGK
Se_KARI VD
VRVG
LRE
-----
GSSS
WKTAEEA
GL
-----
KVTD
MDTAAEEA
D
VIMV
LV
PDEIQPKVYQEHIAAHL
KAGN
Ec_KARI L
DISYA
LRKEAIAEKRAS
WRKATEN
GF
-----
KVGT
YEELIPQA
D
LVIN
LT
PDKQHSDVVR
-
TVQPLM
KDGA
So_KARI V
VVKIG
LRK
-----
GSNS
FAEARAA
GFSEENGT
LGD
MWETISG
SD
LVLL
LIS
DSAQADNYE
-
KVFSHM
KPNS
Os_KARI I
VVKIG
LRK
-----
GSKS
FDEARAA
GFTEESGT
LGD
IWETVSGS
D
LVLL
LI
SDAAQADNYE
-
KIFSHM
KPNS
Features b2 specific. aB b5 aC
b4 aD
Ia_KARI
DLVF
A
H
G
FNIHF
K
---
IIEPPKDSD
VYMIAP
K
S
PG
P
IVRRSYEM
G
-----
GGVP
ALVAVYQ
---
NVSG
Ua_KARI
ALCF
S
H
GFNICFK
---
RIVPPEDV
DVIMVAP
K
A
PGT
EERKAYLE
G
-----
FGVP
GLVAVKQ
---
NPSG
Av_KARI
TLAF
A
H
GFSIHYN
---
QVVPRADL
DVIMIAP
K
A
PGH
TVRSEFVR
G
-----
GGIP
DLIAVYQ
---
D
ASG
Pa_KARI
TLAF
A
H
GFSIHY
N
---
QVVPRADL
DVIMIAP
K
A
PGH
TVRSEFVK
G
-----
GGIP
DLIAIYQ
---
DASG
Aa_KARI
ALAF
A
H
GFNIHF
S
---
QIQPPKDV
DVFMVAP
K
G
PGH
LVRRVYEA
G
-----
GGVP
ALIAVHQ
---
DASG
Se_KARI
TLAF
A
H
GFNIHY
G
---
YIVPPEDV
NVIMCAP
K
G
PGH
IVRRQFTE
G
-----
SGVP
DLACVQQ
---
DATG
Ec_KARI
ALGY
S
H
GFNIVE
V
---
GEQIRKDI
TVVMVAP
K
C
PGT
EVREEYKR
G
-----
FGVP
TLIAV
H
-
PEND
PKG
So_KARI
ILGL
S
H
G
FLLGHLQSL
GQDFPKNI
SVIAVCP
K
GM
G
P
SVRRLYVQGKE
VNGAGI
NSSFAVHQ
---
DVDG
Os_KARI
ILGL
S
H
GFLLGHLQSA
GLDFPKNI
SVIAVCP
K
GM
G
PSVRRLYVQGK
EINGAGIN
SSFAVHQ
---
DVDG
Features b5 m aE b6 m n
aF b7
Ia_KARI E
ALQKALAIAKGI
G
CARA
G
VIE
ST
FKEET
E
T
D
LFG
E
QVILVGGIMELIKASFETLVEE
GY
Ua_KARI
EAREVALAMTKAM
H
WTKA
G
ILE
CT
FEQET
Y
E
D
LFG
E
QCVLCGGLVELMRNGFEVLVEAG
Y
Av_KARI
NAKNLALSYACGV
G
GGRT
G
IIE
TT
FKDET
E
T
D
LFG
E
QAVLCGGCVELVKAGFETLVEA
GY
Pa_KARI
NAKNVALSYACG
V
GGGRTG
IIE
T
TFKDET
E
T
D
LFG
E
QAVLCGGCVELVKAGFETLVEA
GY
Aa_KARI
QAKDLALAYARG
IG
AGRA
G
ILT
T
TFREET
E
T
D
LFG
E
QAVLCGGLSALIKAGFETLVEA
GY
Se_KARI
NAWDIVLSYCWG
V
GGARSG
IIK
A
TFAEET
E
E
D
LFG
E
QAVLCGGLVELVKAGFETLTEA
GY
Ec_KARI
EGMAIAKAWAAA
TG
GHRA
G
VLE
S
S
FVAEV
K
S
D
LMG
E
QTILCGMLQAGSLLCFDKLVEE
GT
So_KARI R
ATDVALGWSIAL
GS
--
P
FTFA
T
T
LEQEYKS
D
I
FG
E
RGILLGAVHGIVECLFRRYTE
SGM
Os_KARI
RATDVALGWSVAL
GS
--
P
FTFA
T
T
LEQEYKS
D
I
FG
E
RGILLGAVHGIVEALFRRYTEQG
M
Features aG b8
h
m a1
Ia_KARI
Q
PEVAYFETVNEL
-
KLIVDLIYEK
G
LTGMLRA
VS
DTAKYGGITVGKFII
D
KSVRDKMKIVLERIRS
G
EFAREWIKEYERG
Ua_KARI
P
PEMAYFECVHEM
-
KLIVDLVWQG
G
IKRMAEV
IS
NTAEYGMWAVGHQI
IG
PEVKEKMKEALKRVE
NG
EFANEWVDEYKR
G
Av_KARI
A
PEMAYFECLHEL
-
KLIVDLMFE
GG
IANMNYS
I
SNNAEYGEYVTGPEVI
N
EQSRQAMRNALKRIQD
G
EYAKMFITEGAA
N
Pa_KARI
APEMAYFECLHEL
-
KLIVDLMYE
GG
IANMNYSI
SN
N
AEYGEYVTGPEVI
N
AESRAAMRNALKRIQ
DG
EYAKMFITEGAA
N
Aa_KARI
QPEIAYFECLHEM
-
KLIVDLIYE
GG
LEYMRYSI
SD
TAQWGDFTSGPRII
N
EETKKEMRRILADIQ
S
GAFAKSWILENQA
N
Se_KARI
PPELAYFECYHEM
-
KMIVDLMYE
SG
IHFMNYS
ISN
TAEYGEYYAGPKVI
N
EQSREAMKEILKRIQ
DG
SFAQEFVDDCN
NG
Ec_KARI
DPAYAEKLIQFGW
-
ETITEALKQ
GG
ITLMMDR
L
SNPAKLRAYALSEQ
-
LKEIMAPLFQKHMDDII
S
GEFSSGMMADWAN
D
So_KARI S
EDLAYKNTVECITGVISKTIST
KG
MLALYNS
LS
EEGKKDFQAAYSAS
-
YYPSMDILYECYEDVAS
G
SEIRSVVLAGRRF
Os_KARI
DEEMAYKNTVEGITGIISKTISK
KG
MLEVYNS
L
TEEGKKEFNKAYSAS
-
FYPCMDILYECYEDVA
SG
SEIRSVVLA
GRR
F
Features
a2
a3
a4 a5
Ia_KARI
---
M
PTVFKELSELEG
S
TIETVGRKLREMMFR
GM
-----
T
FKEET
E
TDLFGEQVILVGGIMELIKASFETLVEE
GY
Ua_KARI
---
I
PFLKASREKMGE
H
QVETVGAEIRKLF
AQHH
-----
T
FEQET
Y
EDLFGEQCVLCGGLVELMRNGFEVLVEAG
Y
Av_KARI
---
Y
PSMTAYRRNNAA
H
QIEVVGEKLRTMM
PWIA
-----
T
FKDET
E
TDLFGEQAVLCGGCVELVKAGFETLVEA
GY
Pa_KARI
---
YPSMTAYRRNNAA
HP
IEQIGEKLRAMM
PWI
------
TFKDET
E
TDLFGEQAVLCGGCVELVKAGFETLVEA
GY
Aa_KARI
---
RPMFNAINRREIE
HP
IEVVGRKLRSM
MPFKAKRPGD
TFREET
E
TDLFGEQAVLCGGLSALIKAGFETLVEA
GY
Se_KARI
---
HKRLLEQREAINT
HP
IETTGAQIRSM
FSWI
------
TFAEET
E
EDLFGEQAVLCGGLVELVKAGFETLTEA
GY
Ec_KARI
---
DKKLLTWREETGK
TA
FETA
PQYEGKI
---------------
GEQEYFDKGVLMIAMVKAGVELAFETMVD
SGI
So_KARI YEKEGLPAFPMGKIDQT
RMWKVGEKVRSV
RPAGDLGPL
------
--------
Y
PFTAGVYVALMMAQIEILRK
KGH
Os_KARI YEKEGLPAFPMGNIDQ
TRMWKVGEKVRSTR
PENDLGPL
------
--------
HPFTAGVYVALMMAQIEVLRKK
GH
Features a6 a7
h
a1+1
Ia_KARI
Q
PEVAYF
E
TVN
E
L
-
KLIVDLIYEK
-
G
LTGMLR
A
VS
DTAKYGGITVGKFII
D
KSVRDKMKIVLERIRS
Ua_KARI
P
PEMAYF
E
CVH
E
M
-
KLIVDLVWQG
-
G
IKRMAE
V
IS
NTAEYGMWAVGHQI
IG
PEVKEKMKEALKRVE
N
Av_KARI
A
PEMAYF
E
CLH
E
L
-
KLIVDLMFE
G
-
G
IANMNY
S
I
SNNAEYGEYVTGPEVI
N
EQSRQAMRNALKRIQD
Pa_KARI
APEMAYF
E
CLH
E
L
-
KLIVDLMYE
G
-
G
IANMNY
S
I
SN
NAEYGEYVTGPEVI
N
AESRAAMRN
ALKRIQ
D
Aa_KARI
QPEIAYF
E
CLH
E
M
-
KLIVDLIYE
G
-
G
LEYMRY
S
I
SD
TAQWGDFTSGPRII
N
EETKKEMRRILADIQ
S
Se_KARI
PPELAYF
E
CYH
E
M
-
KMIVDLMYE
S
-
G
IHFMNY
S
ISN
TAEYGEYYAGPKVI
N
EQSREAMKEILKRIQ
D
Ec_KARI
IEESAYY
E
SLH
E
LPLIANTI
--
AR
KR
LYEMNVVI
SD
TAEYGNYLFSYACVPLLKP
-
FMAE
LQ
P
----
So_KARI SYS
EIIN
E
SVI
E
AVDSLNPFMHAR
-
G
VSFMVDN
C
S
TTARLGSRKWAPRFDYILSQQALVAVDN
----
Os_KARI
SYSEIIN
E
SVI
E
SVDSLNPFMHA
R
-
G
VAFMVDN
C
S
T
TARLGSRKWAPRFDYILTQQAFVTVDK
----
Features
m m a2+1 a3+1
n
n
a4+1
Ia_KARI
G
EFAREWIKEYERG
M
PTVFKELSELE
G
S
TIETVGRKLREMMFR
GM
----------------
Ua_KARI
G
EFANEWVDEYKR
GI
PFLKASREKMGE
H
QVETVGAEIRKLF
AQHH
----------------
Av_KARI
G
EYAKMFITEGAA
N
Y
PSMTAYRRNNAA
H
QIEVVGEKLRTMM
PWIA
----------------
Pa_KARI
G
EYAKMFITEGAA
N
YPSMTAYRRNNAA
HP
IEQIGEKLRAMM
PWI
-----------------
Aa_KARI
GAFAKSWILENQA
N
RPMFNAINRREIE
HP
IEVVGRKLRSM
MPFKAKRPGD
-----------
Se_KARI
G
SFAQEFVDDCN
NG
HKRLLEQREAINT
HP
IETTGAQIRSM
FSWI
-----------------
Ec_KARI GDLGKAIP
-
EGAV
DNGQLRDVNEAIRS
HA
IEQVGKKLRGYMTDMKR
-
IAV
-----------
So_KARI
--------
-----
GAPIN
QDLISNFLS
DP
VHEAIGVCAQL
RPSVDISVTADADFVRPELRQA
Os_KARI
--------
-----
DAPI
NQDLISNFMS
DP
VHGAIEVCAEL
RPTVDIS
---------------
Features
a5+1
a6+1
a7+1
5
Supplemental Information 4: Electron density around the substrate
of Ia_KARI_holo
indicates dehydroxylation of IpoHA. Left: final structure, modelled with
N
-‐
isopropyloxamate (ligand 40E). Right: modelled instead with
N
-‐
hydroxy
-‐
N
-‐
isopropyloxamate (I
poHA; ligand HIO). The
F
o
-‐
F
c
map (red and green) clearly shows
the absence of the
N
-‐
hydroxy moiety. (2F
o
-‐
F
c
map contoured to 1.5
σ
, F
o
-‐
F
c
map
contoured to 4.0
σ
).
6
Supplemental Information 5
: Structural rearrangements around NADPH
nicotinamide in Ia_KARI. Binding of the ni
cotinam
ide
of NADPH (magenta)
to the
the αA
-‐
helix
and
the β6αF
-‐
loop
recruits the
α3
-‐
helix of the other chain of the dimer
(cyan)
.
Ia_KARI_apo
Ia_KARI
_holo
7
Supplemental Information 6
: % Identity between mesophilic Class I KARIs as
determined by
Clustal Omega.
%ID between Av_KARI and:
Ia_KARI
46.9%
Pa_KARI
93.2%
Se_KARI
55.9%
8
Supplemental Information 7
: Amino acid identities and numberings in mesophilic
Class I KARIs
Position in Pa_KARI
Position in Av_KARI
Position in Se_KARI
H107
H107
H118
K130
K130
K141
D190
D190
S201
E194
E194
E205
E226
E226
E237
E230
E230
E241
S249
S249
S260
H134
H134
H145
S26
S26
S37
P132
P132
P143
G133
G133
G144
A131
A131
G142
9
Supplemental Information 8
:
Structural rearrangements around NADPH
nicotinamide
in Av_KARI/Se_KARI. As with Ia_KARI, binding of the nicotinamide of NADPH
(ma
genta) to the
the αA
-‐
helix
and
the β6αF
-‐
loop
recruits the
α
3
-‐
helix of the other
chain of the dimer (cyan)
; in these KARIs more hydrogen bonds are present and
undergo
rearrangements of the hydrogen bond network.
Av_KARI
_met
Se_KARI
_holo
10
Supplemental Information
9
:
Modelling error at P132
-‐
G133 in Pa_KARI
_apo
.
The F
o
-‐
F
c
omit
map (red and green) shows differential density around the peptide bond in
the
published structure (yellow) that is resolved by implementing a 180° peptide
flip (white). This alteration is corroborated by improved backbone hydrogen bond
interactions.
(2F
o
-‐
F
c
map contoured to 1.5
σ
, F
o
-‐
F
c
map contoured to 2.9
σ
).
11
Supplemental Information 10
:
Structural rearrangements around NADPH
nicotinamide in Ec_KARI
Ec_KARI_apo
Ec_KARI
_holo