Martyna−Tobias−Klein · 2015-12-29 · and E164 are located is very flexible allowing for a...
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SUPPLEMENTARY MATERIAL Supplementary material and methods: - Computational details: Molecular Dynamics (MD) simulation was performed for the system CA32F1 to investigate the effect of the introduction of the large side chain of Arg162. After appropriate preparation of the system as explained in the main manuscript an orthorhombic water box with a minimum distance of 10 Å was introduced. The system was then neutralized and 150 mM NaCl added. Equilibration using the default protocol was performed followed by 20 ns NPT simulation at 300 K and 1 atm using DESMOND software.58 with the OPLS- 2005 force field51. The temperature was regulated with the Nosé-Hoover chain thermostat59, with a relaxation time of 1.0 ps, while the pressure was controlled by the Martyna−Tobias−Klein barostat60 with isotropic coupling and a relaxation time of 2.0 ps. The RESPA integrator61 was employed with bonded, near, and far time steps of 2.0, 2.0, and 6.0 fs, respectively. A 9 Å cutoff is used for non-bonded interactions together with the smooth particle mesh Ewald method.62 To minimize the effects of manually introduced mutation V162AR (given the significant change in side chain size) in the model system of CA32F1 we have performed a 20 ns MD simulation. The simulation, shown to stabilize after about 5 ns (Fig. S6) contains an initial orientation of the R162 side chain, placed with Maestro software, that changes along the simulation. Initially, the CZ atom of the guanidino group is about 8 Å away from the carboxylic group of both D205 and E164, as can be seen in Fig. S7. Throughout the simulation it is seen that the loop in which both R162 and E164 are located is very flexible allowing for a diversity of possible arrangements. Two main conformations are observed: one where the R162 is interaction with E164 (between 4 to 11 ns and 13 until 15 ns) and R162 and D205 are between 6 and 11 Å away, and a second where the three amino acids are interacting from 15ns until the end of the simulation. We have selected the last configuration from the MD simulation to use in PELE calculations and have observed that also in PELE simulations this loop is very flexible and all conformations observed in MD are reproduced. Electronic Supplementary Material (ESI) for Catalysis Science & Technology. This journal is © The Royal Society of Chemistry 2015
Transcript of Martyna−Tobias−Klein · 2015-12-29 · and E164 are located is very flexible allowing for a...
SUPPLEMENTARY MATERIAL
Supplementary material and methods:
- Computational details: Molecular Dynamics (MD) simulation was performed for the
system CA32F1 to investigate the effect of the introduction of the large side chain of
Arg162. After appropriate preparation of the system as explained in the main
manuscript an orthorhombic water box with a minimum distance of 10 Å was
introduced. The system was then neutralized and 150 mM NaCl added. Equilibration
using the default protocol was performed followed by 20 ns NPT simulation at 300 K
and 1 atm using DESMOND software.58 with the OPLS- 2005 force field51. The
temperature was regulated with the Nosé-Hoover chain thermostat59, with a relaxation
time of 1.0 ps, while the pressure was controlled by the Martyna−Tobias−Klein
barostat60 with isotropic coupling and a relaxation time of 2.0 ps. The RESPA
integrator61 was employed with bonded, near, and far time steps of 2.0, 2.0, and 6.0 fs,
respectively. A 9 Å cutoff is used for non-bonded interactions together with the smooth
particle mesh Ewald method.62
To minimize the effects of manually introduced mutation V162AR (given the
significant change in side chain size) in the model system of CA32F1 we have
performed a 20 ns MD simulation. The simulation, shown to stabilize after about 5 ns
(Fig. S6) contains an initial orientation of the R162 side chain, placed with Maestro
software, that changes along the simulation. Initially, the CZ atom of the guanidino
group is about 8 Å away from the carboxylic group of both D205 and E164, as can be
seen in Fig. S7. Throughout the simulation it is seen that the loop in which both R162
and E164 are located is very flexible allowing for a diversity of possible arrangements.
Two main conformations are observed: one where the R162 is interaction with E164
(between 4 to 11 ns and 13 until 15 ns) and R162 and D205 are between 6 and 11 Å
away, and a second where the three amino acids are interacting from 15ns until the end
of the simulation. We have selected the last configuration from the MD simulation to
use in PELE calculations and have observed that also in PELE simulations this loop is
very flexible and all conformations observed in MD are reproduced.
Electronic Supplementary Material (ESI) for Catalysis Science & Technology.This journal is © The Royal Society of Chemistry 2015
Supplementary Tables
Table S1. Primers used for mutagenesis of the six residues of the substrate binding
pocket of 3A4 laccase
Primer 5’-3’ sequence
mut162-164-F GCTGCCAAAGTCGGCCCGGCGNNKCCGNNKGCCGATGCTACTCTTATCAAC
mut162-164-R GTTGATAAGAGTAGCATCGGCMNNCGGMNNCGCCGGGCCGACTTTGGCAGC
mut263-264-F CTACTGGATCCGTGCCCTTCCCNNKNNKGGGACCAGGAACTTCGACG
mut263-264-R CGTCGAAGTTCCTGGTCCCMNNMNNGGGAAGGGCACGGATCCAGTAG
mut390-392-F CTCCCCGCCACCTCCGCCGCCNNKGGCNNKCCGCACCCCTTCCACTTG
mut390-392-R CAAGTGGAAGGGGTGCGGMNNGCCMNNGGCGGCGGAGGTGGCGGGGAG
RMLN CCTCTATACTTTAACGTCAAGG
RMLC GGGAGGGCGTGAATGTAAGC
Supplementary Schemes
OH
O
HOOCH3
H3CO
OH
O
OOCH3
H3CO
OH
O
OOCH3
H3CO
HOOCH3
H3CO O
O
OCH3OH
OCH3O
O OOCH3
H3COOCH3
O
OCH3HOO
OHO
OCH3OH
OCH3
OO
O OCH3
H3CO
OCH3OH
OCH3O
O
O
OCH3
H3CO
OCH3O
OCH3
OOCH3
H3CO
HO
OH
OOCH3H3CO
O
LAC + O2
LAC + O2
LAC + O2
Colorless phase( A312)
Red products( A512)
Final product(2,6-dimethoxy-p-benzoquinone)
Scheme S1. Reaction pathway for SA oxidation with the main oxidation intermediates
as proposed by Lacki and Duvnjak (1998). Solid arrows indicate reaction steps
catalyzed by laccase. Dashed arrows indicate non-enzymatic steps.
Supplementary Figures
OH
O
HOOCH3
H3CO
HO
OH
O
OCH3
H3CO
HOOCH3
O
OCH3H3CO
OCH3
O
HOOCH3
H3CO
HO
H3CO
OCH3
HOOCH3
H3CO O
O
OCH3OH
OCH3O
O
A CB
D E F
Figure S1. Chemical structures of SA (A), DAD (B), MS (C), DMP (D), SyA (E) and
MSy (F).
Clone
C1H5
C2F11
C8B12
C11C1
C13E1
C14F12
C18A1
C18C11
C18D11
C30D12 3A
4
Res
idua
l Act
ivity
(%)
0
20
40
60
80
100
120
Figure S2. Thermostability screening assay for selected clones from library C. Initial
activity and residual activity after 10 min incubation at 71 C are shown in white and
striped bars, respectively. Mean values were obtained from five replicates, error bars
represent standard deviations.
Clone
0 500 1000 1500 2000 2500 3000
TAI (
SI 5
12nm
)
0
1
2
3
4Library ALibrary CA
Clone
0 500 1000 1500 2000 2500 3000
TAI (
SI 5
12 n
m)
0
1
2
3
4
Library BLibrary CAB
Figure S3. Activity landscapes for mutant libraries A vs CA (A) and B vs CAB (B).
Figure S4. Hammett (A-C) and Marcus (D-F) plots obtained from competition
reactions with different p-substituted phenols for laccase variants 3A4 (A, D), C14F12
(B, E) and CA32F1 (C, F).
Figure S5. PELE results for the 3A4 (A,B), C14F12 (C,D) and CA32F1 (E,F) variants conformational search of SAH (A,C,E) and SA- (B,D,F).
Figure S6. RMSD vs. time for MD simulation of CA32F1
Figure S7. Plot showing how the distance between R162-E164 and R162-E205 vary
along the MD simulation of CA32F1.
Figure S8. Representative protein-substrate complexes for A) 3A4, B) C14F12 and C) CA32F1 variant with DAD. Each plot represents the best (green) and worse (yellow) position for electron transfer computed with QM/MM.
Figure S9. Representative protein-substrate complexes for A) 3A4 and B) C14F12 and MS. Spin density isosurfaces shown in red and blue.
