SI 1. Construction of 2Ia_KARI-HT and 2Ia_KARI-2L
Inspection of the Ec_KARI structure revealed that the
α
1+1-helix was truncated at its N-terminal
end with respect to the corresponding
α
1’-helix of the Ia_KARI dimer. Therefore, in the third
construct, 2Ia_KARI-HT (helix truncation), we began the duplicate knotted domain at the first
position of this helix which could be aligned to a corresponding position of the Ec_KARI structure,
valine 197. As a result, 2Ia_KARI-HT is eight amino acids shorter than 2Ia_KARI-DD.
Comparing the two knotted domains of Ec_KARI, one final structural difference is clear. The
second knotted domain of Ec_KARI does not have an
α
5-helix, which is replaced by the long
α
4+1
α
6+1-loop (Supplemental Information 2), and the
α
6+1 helix is shifted and rotated with
respect to an alignment with Ia_KARI. Our fourth and final construct, 2Ia_KARI-2L (two
[transplanted] loops), is constructed from 2Ia_KARI-LT by identification of the final positions
which can be aligned around this dissymmetry, lysine 264 and glutamate 315 of Ia_KARI, and
replacing the 50 residues between them, which compose the C-terminal of the
α
4’-helix, the
α
4’
α
5’-loop, the
α
5’-helix, the
α
5’
α
6’-loop, the
α
6’-helix, and the
α
6’
α
7’-loop, with the 44
residues composing the C-terminal of the
α
4+1-helix, the
α
4+1
α
6+1-loop, the
α
6+1helix, and the
α
6+1
α
7+1 loop of Ec_KARI.
However, neither of these constructs yielded sufficient folded, purifiable protein for
characterization.
SI 2. A view of Ec_KARI (PDB 3ULK) showing the two structural deviations from class I KARIs.
The Rossmann domain is shown in green, the first knotted domain is shown in purple, and the second
is shown in pink. The
α
6
α
1+1-loop is shown in orange, connecting the two knotted domains, and the
α
4+1
α
6+1-loop is shown in red.
SI 3. Structural nomenclature for KARIs, from Cahn et al.
28
:
Secondary structure elements are numbered starting from the first
β
-strand that makes up the canonical
Rossmann fold; secondary structure prior to this strand is not conserved. In the Rossmann domain,
β
-strands
are numbered 1–8 and
α
-helices are assigned the letters from A–G. In the knotted domain, which is entirely
α
-helical, helices are assigned numbers from 1 to 8. Loops between secondary structural elements are named
based on the flanking secondary structural elements, as in
β
2
α
B-loop or
α
3
α
4-loop.
In class I KARIs, the secondary structural elements of the dimeric partner are denoted with a prime as in
α
3
′
-
helix. In class II KARIs, the duplicated elements are denoted with ‘+1’, as in
α
3+1-helix.
Ia_KARI -------------------MAKIYKDEDI-SLEPIKNKTIAILGYGSQGRAWALNLRDSGLNVVVGLERQG-----DSWRRAIDD
!
Ec_KARI MANYFNTLNLRQQLAQLGKCRFMGRDEFADGASYLQGKKVVIVGCGAQGLNQGLNMRDSGLDISYALRKEAIAEKRASWRKATEN
!
2Ia_KARI-DD -------------------MAKIYKDEDI-SLEPIKNKTIAILGYGSQGRAWALNLRDSGLNVVVGLERQG-----DSWRRAIDD
!
2Ia_KARI-HT -------------------MAKIYKDEDI-SLEPIKNKTIAILGYGSQGRAWALNLRDSGLNVVVGLERQG-----DSWRRAIDD
!
2Ia_KARI-LT -------------------MAKIYKDEDI-SLEPIKNKTIAILGYGSQGRAWALNLRDSGLNVVVGLERQG-----DSWRRAIDD
!
2Ia_KARI-2L -------------------MAKIYKDEDI-SLEPIKNKTIAILGYGSQGRAWALNLRDSGLNVVVGLERQG-----DSWRRAIDD
!
!
Ia_KARI GFKPMYTKDAVAIADIIVFLVPDMVQKSLWLNSVKDFMKKGADLVFAHGFNIHFKIIEPPKDSDVYMIAPKSPGPIVRRSYEMGG
!
Ec_KARI GFKVGTYEELIPQADLVINLTPDK-QHSDVVRTVQPLMKDGAALGYSHGFNIVEVGEQIRKDITVVMVAPKCPGTEVREEYKRGF
!
2Ia_KARI-DD GFKPMYTKDAVAIADIIVFLVPDMVQKSLWLNSVKDFMKKGADLVFAHGFNIHFKIIEPPKDSDVYMIAPKSPGPIVRRSYEMGG
!
2Ia_KARI-HT GFKPMYTKDAVAIADIIVFLVPDMVQKSLWLNSVKDFMKKGADLVFAHGFNIHFKIIEPPKDSDVYMIAPKSPGPIVRRSYEMGG
!
2Ia_KARI-LT GFKPMYTKDAVAIADIIVFLVPDMVQKSLWLNSVKDFMKKGADLVFAHGFNIHFKIIEPPKDSDVYMIAPKSPGPIVRRSYEMGG
!
2Ia_KARI-2L GFKPMYTKDAVAIADIIVFLVPDMVQKSLWLNSVKDFMKKGADLVFAHGFNIHFKIIEPPKDSDVYMIAPKSPGPIVRRSYEMGG
!
!
Ia_KARI GVPALVAVY--QNVSGEALQKALAIAKGIGCARAGVIESTFKEETETDLFGEQVILVGGIMELIKASFETLVEEGYQPEVAYFET
!
Ec_KARI GVPTLIAVHPENDPKGEGMAIAKAWAAATGGHRAGVLESSFVAEVKSDLMGEQTILCGMLQAGSLLCFDKLVEEGTDPAYAEKLI
!
2Ia_KARI-DD GVPALVAVY--QNVSGEALQKALAIAKGIGCARAGVIESTFKEETETDLFGEQVILVGGIMELIKASFETLVEEGYQPEVAYFET
!
2Ia_KARI-HT GVPALVAVY--QNVSGEALQKALAIAKGIGCARAGVIESTFKEETETDLFGEQVILVGGIMELIKASFETLVEEGYQPEVAYFET
!
2Ia_KARI-LT GVPALVAVY--QNVSGEALQKALAIAKGIGCARAGVIESTFKEETETDLFGEQVILVGGIMELIKASFETLVEEGYQPEVAYFET
!
2Ia_KARI-2L GVPALVAVY--QNVSGEALQKALAIAKGIGCARAGVIESTFKEETETDLFGEQVILVGGIMELIKASFETLVEEGYQPEVAYFET
!
!
Ia_KARI VNELKLIVDLIYEKGLTGMLRAVSDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELEGST
!
Ec_KARI QFGWETITEALKQGGITLMMDRLSNPAKLRAYALSEQ-LKEIMAPLFQKHMDDIISGEFSSGMMADWANDDKKLLTWREETGKTA
!
2Ia_KARI-DD VNELKLIVDLIYEKGLTGMLRAVSDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELEGST
!
2Ia_KARI-HT VNELKLIVDLIYEKGLTGMLRAVSDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELEGST
!
2Ia_KARI-LT VNELKLIVDLIYEKGLTGMLRAVSDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELE
KTA
!
2Ia_KARI-2L VNELKLIVDLIYEKGLTGMLRAVSDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELE
KTA
!
!
Ia_KARI IETVGRKLREMMFRGMKQISSH---------------------------------------------------------------
!
Ec_KARI FETAPQYEGKI---GEQEYFDKG-------VLMIAMVKAGVELAFETMVDSGIIEESAYYESLHELPLIANTIARKRLYEMNVVI
!
2Ia_KARI-DD IETVGRKLREMMFRGMKQISSHETDLFGEQVILVGGIMELIKASFETLVEEGYQPEVAYFETVNELKLIVDLIYEKGLTGMLRAV
!
2Ia_KARI-HT IETVGRKLREMMFRGMKQISSH-
-------
VILVGGIMELIKASFETLVEEGYQPEVAYFETVNELKLIVDLIYEKGLTGMLRAV
!
2Ia_KARI-LT
FETAPQYEGKI---GEQEYFDKGV------
VILVGGIMELIKASFETLVEEGYQPEVAYFETVNELKLIVDLIYEKGLTGMLRAV
!
2Ia_KARI-2L
FETAPQYEGKI---GEQEYFDKGV------
VILVGGIMELIKASFETLVEEGYQPEVAYFETVNELKLIVDLIYEKGLTGMLRAV
!
!
Ia_KARI -------------------------------------------------------------------------------------
!
Ec_KARI SDTAEYGNYLFSYACVPL-----LKPFMAELQPGDLGKA-IPEGAVDNGQLRDVNEAIRSHAIEQVGKKLRGYM-TDMKRIAVAG
!
2Ia_KARI-DD SDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELEGSTIETVGRKLREMMFRGMKQISSH-
!
2Ia_KARI-HT SDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELEGSTIETVGRKLREMMFRGMKQISSH-
!
2Ia_KARI-LT SDTAKYGGITVGKFIIDKSVRDKMKIVLERIRSGEFAREWIKEYERGMPTVFKELSELEGSTIETVGRKLREMMFRGMKQISSH-
!
2Ia_KARI-2L SDTAKYGGITVGK
ACVPL-----LKPFMAELQPGDLGKA-IPEGAVDNGQLRDVNEAIRSHA
IETVGRKLREMMFRGMKQISSH-
!
SI 4. An alignment of the sequences of Ia_KARI, Ec_KARI, and the four designed 2Ia_KARI constructs.
Red text in the 2Ia_KARI constructs shows sequence insertions or deletions coming from Ec_KARI.
!
Enzyme
K
M
NADH
[
μ
M]
K
M
NADPH
[
μ
M]
k
cat
NADH
[s
-
1
]
k
cat
NADPH
[s
-
1
]
k
cat
/
K
M
NADH
[s
-
1
mM
-
1
]
k
cat
/
K
M
NADPH
[s
-
1
mM
-
1
]
Catalytic
Efficiency Ratio
(NADH/NADPH)
Ia_KARI
< 1
< 1
2.6
3.3
>2,600
>3,300
0.8
2Ia_KARI
-
DD
< 1
< 1
1.7
3.0
>1,700
>3,000
0.6
2Ia_KARI
-
LT
< 1
< 1
0.8
1.0
>800
>1,000
0.8
SI 5. Comparison between catalytic parameters on NADH and NADPH for selected KARIs.
-‐100
0
100
200
300
400
500
600
700
800
900
1000
0
2
4
6
8
10
12
14
16
Absorbance at 280 nm (mAU)
Reten4on Volume (mL)
Ia_KARI
2Ia_KARI-‐DD
2Ia_KARI-‐LT
BSA
Myoglobin
Ia_KARI
2Ia_KARI-‐
DD
2Ia_KARI-‐LT
BSA
Myoglobin
5
7
9
11
13
15
4
4.2
4.4
4.6
4.8
5
Peak Reten4on Volume (mL)
Log(Predicted Molecular Weight)
SI 6. Gel filtration chromatography analysis of 2Ia_KARIs. Gel filtration curves are shown for 2Ia_KARI-
DD and 2Ia-KARI-LT, along with dimeric Ia_KARI WT and flanking controls (BSA and myoglobin). Inset
shows proper power-law correspondence for predicted monomeric molecular weight.
R² = 0.99853
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Enzyme ac4vity (arbitrary units)
Protein Concentra4on (mg/mL)
0.00
0.05
0.10
0.15
0.20
0.00
0.01
0.02
SI 7. Linear relationship between enzyme concentration and activity across a 512-fold range of dilutions for
2Ia_KARI-DD, showing monomeric behavior.
Data CollecGon
Space Group
P4
3
22
a, b, c (Å)
103.9, 103.9, 142.1
α, β, γ (°)
90, 90, 90
ResoluGon (Å)
103.9 – 1.94 (1.99-‐1.94)
R
p.i.m
(%)
2.6 (22.2)
<i>/<σi>
7.39 (0.74)
Completeness (%)
99.2 (94.4)
Redundancy
10.3 (8.2)
Refinement
No. reflecGons
Rwork/Rfree (%)
15.8/17.9 (21.8/27.1)
No. atoms
Protein
3753
Ligand
68
Water
237
RMSD
Bond lengths (Å)
0.020
Bond angles (°)
1.128
Ramachandran Plot
Favored
466
Allowed
14
Outliers
0
SI 8. Selected statistics on data collection and structure refinement for the
structure of 2Ia_KARI-DD (RCSB 5E4R). Values in parentheses refer to the
highest resolution shell.
SI 9. The packing of the obtained crystal of 2Ia_KARI-DD. A single protein chain (cyan and magenta)
composes the asymmetric unit, and symmetry mates are shown in grey. Spheres represent the residues
flanking the structurally unresolved interdomain connector.
0
2
4
6
8
10
12
14
0
40
80
120
160
200
240
280
320
360
400
440
480
Cα-‐Cα distance (Å)
Residue number
SI 10. The C
α
-C
α
distances between 2Ia_KARI-DD and Ia_KARI. The lines are colored by which monomer
of Ia_KARI is closer, blue for the first monomer and green for the second. When multiple C
α
are present for
a given residue, due to alternate conformations, the greater distance is shown in a lighter color. The residues
between 330 and 339 are not structurally resolved.
Rossmann domain
first knoied domain
second knoied domain
SI 11. The
α
5+1-helix, showing two distinct conformations
visible in the 2Fo-Fc map, contoured to 1.2
σ
(blue) and
0.65
σ
(cyan).