JBC Papers in Press. Published on September 29, 2014 as Manuscript M114.567008 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.567008 R-modules in alginate epimerases
Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii Edith Buchinger1,2, Daniel H. Knudsen1, Manja A. Behrens3, Jan Skov Pedersen3, Olav A. Aarstad2, Anne Tøndervik4, Svein Valla3, Gudmund Skjåk-Bræk2, Reinhard Wimmer1 and Finn L. Aachmann2,* 1
Department of Biotechnology, Chemistry and Environmental Engineering, Sohngaardsholmsvej 49, DK9000 Aalborg , Denmark. 2
NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
3
4
Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000, Denmark.
Department of Bioprocess technology, SINTEF Materials and Chemistry, N-7465 Trondheim, Norway
To whom correspondence should be addressed: Finn L. Aachmann, NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway, Tel.:+4773593317; Fax: +4773591283; E-mail: [email protected] Keywords: Alginate, Alginate C-5 Epimerase, R-module, NMR, ITC Background: Alginate epimerases consist of NMR structure of the three individual Rcatalytic and non-catalytic domains of yet modules from AlgE6 (AR1R2R3) and the unknown function. overall structure of both AlgE4 (AR) and Results: The non-catalytic domains of AlgE4 and AlgE6 using Small angle X-ray Scattering AlgE6 possess different alginate binding (SAXS). Furthermore, the alginate binding behaviour despite highly similar structures. ability of the R-modules of AlgE4 and AlgE6 Conclusion: Non-catalytic subunits of AlgE6 and has been studied with NMR and ITC. The AlgE4 influence the product specificity of the AlgE6 R-modules fold into an elongated catalytic domain. parallel -roll with a shallow, positively Significance: The work opens a new route to charged groove across the module. SAXS designing alginate epimerases producing tailored analyses of AlgE4 and AlgE6 show an overall alginates. elongated shape with some degree of flexibility between the modules for both enzymes. Abstract Titration of the R-modules with defined The bacterium Azotobacter vinelandii alginate oligomers show strong interaction produces a family of seven secreted and between AlgE4R and both oligo-M and MG, calcium-dependent mannuronan C-5 while no interaction was detected between these epimerases (AlgE1-7). These epimerases are oligomers and the individual R-modules from responsible for the epimerization of β-DAlgE6. A combination of all three R-modules mannuronic acid (M) to α-L-guluronic acid (G) from AlgE6 shows weak interaction with long in alginate polymers. The epimerases display a M-oligomers. Exchanging the R-modules modular structure being composed of one or between AlgE4 and AlgE6 resulted in a novel two catalytic A-modules and from one to seven epimerase called AlgE64 with increased GR-modules having an activating effect on the Ablock forming ability compared to AlgE6. module. In this study we have determined the ________________________________________
1 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
*Running title: R-modules in alginate epimerases
R-modules in alginate epimerases
M residues are arranged in pattern showing homopolymeric regions of G residues (Gblocks) and homopolymeric regions of M residues (M-blocks) interspersed by regions in which the two groups coexist in a strictly alternating sequence (MG-blocks). The wide variability of the composition and sequential structure of alginate is a key functional attribute of the polysaccharide. While the intrinsic inflexibility of the alginate chain increases in the order MG 100 Hz are plotted as a dot along the primary amino acid sequnce for the alginate oligomers that were used in the experiments. When more than one amino acid in a row experience chemical shift changes it results in bar, where the length is proportional to the affected area. The alginate oligomer type and length has hardly any influence on the location of the perturbation. B. Amino acid residues with a chemical shift change |δbound| > 100 Hz for the interaction with hexameric M are highlighted in blue on the backbone ribbon structure of the AlgE4R-modul
Fig. 8: Charged amino acids on the assumed alginate binding surface. A. AlgE4R, B. AlgE6R1, C. AlgE6R2 and D. AlgE6R3. Each R-module has 5-7 basic amino acids (in blue) and 5-7 acidic amino acids (in red) within the assumed alginate binding region of the surface. AlgE4R has the more basic amino acid residues evenly distributed over the assumed alginate binding surface then the R-modules of AlgE6.
Tables Tab. 1: Plasmids used in this study.
Plasmid
Description
pTYB12
IMPACT-CN fusion vector containing an N-terminal New England
pTYB4
Reference
chitin-binding-tag
Biolab
IMPACT-CN fusion vector containing a C-terminal
New England
chitin-binding-tag
Biolabobtained from NEB
pFA8
Derivate of pTYB12 in which the DNA sequence of (23) AlgE6R1 was inserted as a BsmI-XmaI fragment
pFA12
Derivate of pTYB12 in which the DNA sequence of (24)
19
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
Fig. 7: Results for interaction between R-module and alginate obtained with ITC and NMR. The graph shows Kd obtained from the ITC (A) and NMR (B) measurements. In general, MG oligomers have higher dissociation constants than poly-M at the same degree of polymerisation. NMR data fit well to the data obtained by ITC. Kd depends strongly on the degree of polymerisation.
R-modules in alginate epimerases
AlgE6R2 was inserted as a BsmI-XmaI fragment pFA13
Derivative of pTYB12 in which the DNA sequence of (25) AlgE6R3 was inserted as a BsmI -XmaI fragment
pTrc99A
Expression vector
(56)
pMV3
Pm/XylS expression vector containing a mutation in Pm (57) leading to increased expression levels
pAT116
A synthetic DNA sequence encoding the A-module from This study AlgE4 combined with the R-modules from AlgE6
XmaI-EcoRI fragment pAT117
A synthetic DNA sequence encoding the A-module from This study and AlgE6 combined with the R-module from AlgE4 (22) (AlgE6ARAlgE4) was inserted into pMV3 as an NdeI-NotI fragment
pAT118
A synthetic DNA sequence encoding the R-modules from This study AlgE6 (R1R2R3) was inserted into pTYB12 as an EcoRI-NruI fragment
pBS32
Derivative of pTYB4 in which the DNA sequence of unpublished AlgE4 was inserted as a NcoI-XmaI fragment
pFA1
results.
Derivate of pTYB11 opened with SapI and treated with (58) PeaR7I before insertion of the DNA sequence of AlgE4R
pTB26
Derivative of pTYB4 in which the DNA sequence of (13) AlgE4A was inserted as a NcoI-XmaI fragment
pMD4
Derivative of pTYB12 in which the DNA sequence of (53)
20
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
(AlgE4A(R1R2R3)AlgE6)was inserted into pTrc99A as an
R-modules in alginate epimerases
ORF-9. Bacterial strains DH5α
Escherichia coli DH1 derivate with deoR, nupG, (59) 80dlacZM15 and (lacZY-argF)U169
ER2566
F--fhuA2 [Ion] ompT lac::T7gene1 gal SulA11 (mcrC- New England mrr)114::IS10
R(mcr-73::miniTn10-TetS)2R(zgb- Biolab
210::Tn10) (TetS) endA1 [dcm] XL10-Gold
endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Stratagene
lacIqZΔM15 Tn10(TetR Amy CmR)] RV308
lacIq-, su-, ΔlacX74, gal IS II::OP308, strA
21
ATCC31608
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tetR F'[proAB
R-modules in alginate epimerases
Tab. 3: Results obtained from the SAXS data by performing an IFT analysis. Molecular weight was calculated from I(q = 0). The calculated molecular weights were determined by use of ProtParam (60). Protein AlgE4 AlgE4A AlgE4R AlgE6 AlgE6R1 AlgE6R2 AlgE6R3
Rg (IFT analysis)[Å] 31.3±1 22.0±1 18.7±1 55.4±1 15.6±1 16.0±1 16.6±1
Rg (Guinier analysis) [Å] 31.1±1 20.1±1 19.4±1 51.8±1 16.3±1 16.5±1 16.8±1
22
Dmax [Å]
MwI(0) [kDa]
Mw [kDa]
100±10 65±10 50±5 180±10 45±5 47±5 50±5
53±6 44±5 17±2 100±10 16±1.5 20±2 21±3
57.7 39.9 17.0 90.2 15.5 16.5 18.2
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
Tab. 2: Summary of the results of the structure calculations of the three R-module structures. The structure of each module was calculated with and without Ca2+-ion incorporation. All RMSDs calculations were calculated in MolMol (61). AlgE6R1 AlgE6R2 AlgE6R3 Without With Without With Without With Ca2+-ions Ca2+-ions Ca2+-ions Ca2+-ions Ca2+-ions Ca2+-ions Total number of NOE 1517 3554 3535 3579 2057 2146 constraints Intra 650 2181 2316 2316 865 889 Short 476 782 760 760 589 606 Medium 42 112 106 106 118 98 Long 349 479 353 397 485 553 Cyana target function 2.81 ± 1.18 2.55 ± 1.17 ± 0.30 2.65 ± 2.70 ± 0.86 1.17 ± value (Å) 0.92 0.4 0.42 Distance constraint < 0.1 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 violation ( > 10 structures) RMSD 1.651 2.397 2.256 1.967 1.769 1.864 N, Cα, C’ (core-Rmodule a) N, Cα, C’ (secondary 0.671 1.178 1.191 1.038 0.954 0.941 structure elements b) Heavy Atoms (core R1.945 2.840 2.732 2.455 2.015 2.077 module a) Heavy Atoms (secondary 1.163 1.933 1.649 1.521 1.379 1.354 structure elements b) a Core-R-modules are from the first identical amino (G1 in AlgE4R, G3 in AlgE6R1, G11 in AlgE6R2 and G11 in AlgE6R3 see also Fig. S1) acid until the C-terminal end of AlgE6R1 and AlgE6R2 or the amino acid that is in the same position when the 4 structures are aligned (T150 in AlgE4R and A162 in AlgE6R3). b The secondary structure elements are only the β-strands (Fig. 1 and 2)
R-modules in alginate epimerases Tab. 4: Dissociation constants and other thermodynamic data for the R-module from AlgE4 determined at 25C. Calculations from NMR data were done assuming a 1:1 (N = 1) complex. Nearly all the ITC data could be fitted to N = 1.M8 and M9 could not be fitted with one binding event. Data obtained by calculating two binding events are indicated by #. NMR
ITC
Ligand
Kd[µM]
N
Kd[µM]
ΔH [kJ/mol] -ΔST[kJ/mol]
M3
883 ± 98.1
Fixed to 1
323 ± 3.8
-33.8 ± 0.2
-13.9
-19.9 ± 1.4
M4
191 ± 27.2
Fixed to 1
26 ± 2.0
-45.4 ± 1.4
-19.3
-26.1 ± 2.4
M5
2.72 ± 1.91 Fixed to 1
3.75 ± 0.06 -41.9 ± 0.2
-10.9
-31.0 ± 1.1
M6
0.41 ± 0.30 Fixed to 1
1.03 ± 0.25 -46.8 ± 1.7
-12.6
-34.2 ± 2.1
M7
---
Fixed to 1
0.46 ±0.02
-45.0 ± 0.2
-8.8
-36.2 ± 0.9
M8 -1 #
---
0.499 ± 0.016 0.36 ± 0.02 -44.1 ± 3.1
-7.4
-36.7 ± 3.2
M8 - 2 # ---
0.465 ± 0.017 0.09 ± 0.01 -83.2 ± 2.2
-42.8
-40.4 ± 4.8
M9 - 1 # ---
0.418 ± 0.018 0.57 ± 0.03 -11.6 ± 6.3
24.0
-35.6 ± 6.7
M9 - 2 # ---
0.601 ± 0.02
0.15 ± 0.02 -91.8 ± 1.8
-52.9
-38.9 ± 5.6
MG4
156 ± 23.4
Fixed to 1
2040 ± 110 -47.8 ± 1.55
-32.4
-15.4 ± 3.6
MG5
---
Fixed to 1
4115 ± 239 -230 ± 12.8
-216.1
-13.9 ± 25.2
MG6
---
Fixed to 1
1204 ± 67
-98.5 ± 2.99
-81.9
-16.6 ± 8.7
MG7
314 ± 75.2
---
---
---
---
---
MG8
---
Fixed to 1
1330 ± 132 -210 ± 20.1
-652.1
-15.9 ± 27.9
ΔG [kJ/mol]
23
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
Tab. 5: Distribution of M and G units obtained from endpoint epimerisation of poly-M. Monomer, diades, triades and average G-block length calculated based on 1H NMR spectra obtained from poly-M epimerised with AlgE46 or AlgE64. FG FM FGG FMG/GM FMM FMGG/GGM FMGM FGGG NG>1 AlgE4 0.46 0.54 0 0.46 0.08 0 0.46 0 0 AlgE46 0.41 0.59 0 0.41 0.17 0 0.41 0 0 AlgE6 0.62 0.38 0.52 0.11 0.272 0.03 0.09 0.52 12.0 AlgE64 0.82 0.18 0.75 0.08 0.10 0.06 0.07 0.69 16.0
Figure 1
----------GSD-GEPLVGGDTDDQLQGGSGADRLDGGAGDDILDGGAGRDRLSGGAGA ---------QGTDGNDVLIGSDVGEQISGGAGDDRLDGGAGDDLLDGGAGRDRLTGGLGA DPSAEAQPIVGSDLDDQLHGTLLGEEISGGGGADQLYGYGGGDLLDGGAGRDRLTGGEGA DPGVEGTPVVGSDLDDELHGTLGSEQILGGGGADQLYGYAGNDLLDGGAGRDKLSGGEGA *:* .: * * .::: **.* *:* * .*.*:********:*:** ** DTFVFSAREDSYRTDTAVFNDLILDFEASEDRIDLSALGFSGLGDGYGGTLLLKTNAEGT DTFRFALREDSHRSPLGTFSDLILDFDPSQDKIDVSALGFIGLGNGYAGTLAVSLSADGL DTFRFALREDSQRSAAGTFSDLILDFDPTQDKLDVSALGFTGLGNGYAGTLAVSVSDDGT DTFRFALREDSHRSPLGTFGDRILDFDPSQDRIDVSALGFSGLGNGYAGSLAVSVSDDGT *** *: **** *: ..*.* ****:.::*::*:***** ***:**.*:* :. . :* RTYLKSFEADAEGRRFEVALDGDHTGDLSAANVVFAATGTTT--ELEVLGDSGTQAGAIV RTYLKSYDADAQGRSFELALDGNHAATLSAGNIVFAAATPG------------------RTYLKSYETDAEGRSFEVSLQGNHAAALSADNILFATPVPV------------------RTYLKSYEADAQGLSFEVALEGDHAAALSADNIVFAATDAAAAGELGVIGASGQPDDPTV ******:::**:* **::*:*:*:. *** *::**:. .
AlgE4R AlgE6R1 AlgE6R2 AlgE6R3
AlgE4R AlgE6R1 AlgE6R2 AlgE6R3
AlgE4R AlgE6R1 AlgE6R2 AlgE6R3
167 152 161 180
109 111 120 120
49 51 60 60
Figure 2:
Figure 3
B.
D.
A.
C.
Figure 4
Figure 5
0 9.70
20
40
60
80
100
120
Relative height
9.60
9.55 Chemical Shift [ppm]
9.65
9.50
9.45
Figure 6
A. B.
Figure 7
Figure 8
D15
C.
A.
R51
R53
D26
R24
R42
R40
D85
K103
D94
E128
D130
K125
R121 D118
E137
R73
D74
R114
E117
R110 D130 E107
E126
R62
R124
D.
E17
D15
D28
E128
R64
K53
R51 R82
D85
E141
R121
K125
D109
K116
E138
R73
D132 R112 D76
R44
D24 R42
R26
B.
D119
D118
Protein Structure and Folding: Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii Edith Buchinger, Daniel H. Knudsen, Manja A. Behrens, Jan Skov Pedersen, Olav A. Aarstad, Anne Tøndervik, Svein Valla, Gudmund Skjåk-Braek, Reinhard Wimmer and Finn L. Aachmann
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J. Biol. Chem. published online September 29, 2014
Structural and Functional Characterization of the R-modules in Alginate C-5 Epimerases AlgE4 and AlgE6 from Azotobacter vinelandii Edith Buchinger1,2, Daniel H. Knudsen1, Manja A. Behrens3, Jan Skov Pedersen3, Olav A. Aarstad2, Anne Tøndervik4, Svein Valla3, Gudmund Skjåk-Bræk2, Reinhard Wimmer1 and Finn L. Aachmann2,* 1
Department of Biotechnology, Chemistry and Environmental Engineering, Sohngaardsholmsvej 49, DK9000 Aalborg , Denmark. 2
NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway.
3
4
Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, Gustav Wieds Vej 14, DK-8000, Denmark.
Department of Bioprocess technology, SINTEF Materials and Chemistry, N-7465 Trondheim, Norway
To whom correspondence should be addressed: Finn L. Aachmann, NOBIPOL, Department of Biotechnology, Norwegian University of Science and Technology, Sem Sælands vei 6/8, 7491 Trondheim, Norway, Tel.:+4773593317; Fax: +4773591283; E-mail: [email protected] Keywords: Alginate, Alginate C-5 Epimerase, R-module, NMR, ITC Background: Alginate epimerases consist of NMR structure of the three individual Rcatalytic and non-catalytic domains of yet modules from AlgE6 (AR1R2R3) and the unknown function. overall structure of both AlgE4 (AR) and Results: The non-catalytic domains of AlgE4 and AlgE6 using Small angle X-ray Scattering AlgE6 possess different alginate binding (SAXS). Furthermore, the alginate binding behaviour despite highly similar structures. ability of the R-modules of AlgE4 and AlgE6 Conclusion: Non-catalytic subunits of AlgE6 and has been studied with NMR and ITC. The AlgE4 influence the product specificity of the AlgE6 R-modules fold into an elongated catalytic domain. parallel -roll with a shallow, positively Significance: The work opens a new route to charged groove across the module. SAXS designing alginate epimerases producing tailored analyses of AlgE4 and AlgE6 show an overall alginates. elongated shape with some degree of flexibility between the modules for both enzymes. Abstract Titration of the R-modules with defined The bacterium Azotobacter vinelandii alginate oligomers show strong interaction produces a family of seven secreted and between AlgE4R and both oligo-M and MG, calcium-dependent mannuronan C-5 while no interaction was detected between these epimerases (AlgE1-7). These epimerases are oligomers and the individual R-modules from responsible for the epimerization of β-DAlgE6. A combination of all three R-modules mannuronic acid (M) to α-L-guluronic acid (G) from AlgE6 shows weak interaction with long in alginate polymers. The epimerases display a M-oligomers. Exchanging the R-modules modular structure being composed of one or between AlgE4 and AlgE6 resulted in a novel two catalytic A-modules and from one to seven epimerase called AlgE64 with increased GR-modules having an activating effect on the Ablock forming ability compared to AlgE6. module. In this study we have determined the ________________________________________
1 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
*Running title: R-modules in alginate epimerases
R-modules in alginate epimerases
M residues are arranged in pattern showing homopolymeric regions of G residues (Gblocks) and homopolymeric regions of M residues (M-blocks) interspersed by regions in which the two groups coexist in a strictly alternating sequence (MG-blocks). The wide variability of the composition and sequential structure of alginate is a key functional attribute of the polysaccharide. While the intrinsic inflexibility of the alginate chain increases in the order MG 100 Hz are plotted as a dot along the primary amino acid sequnce for the alginate oligomers that were used in the experiments. When more than one amino acid in a row experience chemical shift changes it results in bar, where the length is proportional to the affected area. The alginate oligomer type and length has hardly any influence on the location of the perturbation. B. Amino acid residues with a chemical shift change |δbound| > 100 Hz for the interaction with hexameric M are highlighted in blue on the backbone ribbon structure of the AlgE4R-modul
Fig. 8: Charged amino acids on the assumed alginate binding surface. A. AlgE4R, B. AlgE6R1, C. AlgE6R2 and D. AlgE6R3. Each R-module has 5-7 basic amino acids (in blue) and 5-7 acidic amino acids (in red) within the assumed alginate binding region of the surface. AlgE4R has the more basic amino acid residues evenly distributed over the assumed alginate binding surface then the R-modules of AlgE6.
Tables Tab. 1: Plasmids used in this study.
Plasmid
Description
pTYB12
IMPACT-CN fusion vector containing an N-terminal New England
pTYB4
Reference
chitin-binding-tag
Biolab
IMPACT-CN fusion vector containing a C-terminal
New England
chitin-binding-tag
Biolabobtained from NEB
pFA8
Derivate of pTYB12 in which the DNA sequence of (23) AlgE6R1 was inserted as a BsmI-XmaI fragment
pFA12
Derivate of pTYB12 in which the DNA sequence of (24)
19
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
Fig. 7: Results for interaction between R-module and alginate obtained with ITC and NMR. The graph shows Kd obtained from the ITC (A) and NMR (B) measurements. In general, MG oligomers have higher dissociation constants than poly-M at the same degree of polymerisation. NMR data fit well to the data obtained by ITC. Kd depends strongly on the degree of polymerisation.
R-modules in alginate epimerases
AlgE6R2 was inserted as a BsmI-XmaI fragment pFA13
Derivative of pTYB12 in which the DNA sequence of (25) AlgE6R3 was inserted as a BsmI -XmaI fragment
pTrc99A
Expression vector
(56)
pMV3
Pm/XylS expression vector containing a mutation in Pm (57) leading to increased expression levels
pAT116
A synthetic DNA sequence encoding the A-module from This study AlgE4 combined with the R-modules from AlgE6
XmaI-EcoRI fragment pAT117
A synthetic DNA sequence encoding the A-module from This study and AlgE6 combined with the R-module from AlgE4 (22) (AlgE6ARAlgE4) was inserted into pMV3 as an NdeI-NotI fragment
pAT118
A synthetic DNA sequence encoding the R-modules from This study AlgE6 (R1R2R3) was inserted into pTYB12 as an EcoRI-NruI fragment
pBS32
Derivative of pTYB4 in which the DNA sequence of unpublished AlgE4 was inserted as a NcoI-XmaI fragment
pFA1
results.
Derivate of pTYB11 opened with SapI and treated with (58) PeaR7I before insertion of the DNA sequence of AlgE4R
pTB26
Derivative of pTYB4 in which the DNA sequence of (13) AlgE4A was inserted as a NcoI-XmaI fragment
pMD4
Derivative of pTYB12 in which the DNA sequence of (53)
20
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
(AlgE4A(R1R2R3)AlgE6)was inserted into pTrc99A as an
R-modules in alginate epimerases
ORF-9. Bacterial strains DH5α
Escherichia coli DH1 derivate with deoR, nupG, (59) 80dlacZM15 and (lacZY-argF)U169
ER2566
F--fhuA2 [Ion] ompT lac::T7gene1 gal SulA11 (mcrC- New England mrr)114::IS10
R(mcr-73::miniTn10-TetS)2R(zgb- Biolab
210::Tn10) (TetS) endA1 [dcm] XL10-Gold
endA1 glnV44 recA1 thi-1 gyrA96 relA1 lac Hte Stratagene
lacIqZΔM15 Tn10(TetR Amy CmR)] RV308
lacIq-, su-, ΔlacX74, gal IS II::OP308, strA
21
ATCC31608
Downloaded from http://www.jbc.org/ by guest on March 9, 2015
Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 tetR F'[proAB
R-modules in alginate epimerases
Tab. 3: Results obtained from the SAXS data by performing an IFT analysis. Molecular weight was calculated from I(q = 0). The calculated molecular weights were determined by use of ProtParam (60). Protein AlgE4 AlgE4A AlgE4R AlgE6 AlgE6R1 AlgE6R2 AlgE6R3
Rg (IFT analysis)[Å] 31.3±1 22.0±1 18.7±1 55.4±1 15.6±1 16.0±1 16.6±1
Rg (Guinier analysis) [Å] 31.1±1 20.1±1 19.4±1 51.8±1 16.3±1 16.5±1 16.8±1
22
Dmax [Å]
MwI(0) [kDa]
Mw [kDa]
100±10 65±10 50±5 180±10 45±5 47±5 50±5
53±6 44±5 17±2 100±10 16±1.5 20±2 21±3
57.7 39.9 17.0 90.2 15.5 16.5 18.2
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Tab. 2: Summary of the results of the structure calculations of the three R-module structures. The structure of each module was calculated with and without Ca2+-ion incorporation. All RMSDs calculations were calculated in MolMol (61). AlgE6R1 AlgE6R2 AlgE6R3 Without With Without With Without With Ca2+-ions Ca2+-ions Ca2+-ions Ca2+-ions Ca2+-ions Ca2+-ions Total number of NOE 1517 3554 3535 3579 2057 2146 constraints Intra 650 2181 2316 2316 865 889 Short 476 782 760 760 589 606 Medium 42 112 106 106 118 98 Long 349 479 353 397 485 553 Cyana target function 2.81 ± 1.18 2.55 ± 1.17 ± 0.30 2.65 ± 2.70 ± 0.86 1.17 ± value (Å) 0.92 0.4 0.42 Distance constraint < 0.1 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 violation ( > 10 structures) RMSD 1.651 2.397 2.256 1.967 1.769 1.864 N, Cα, C’ (core-Rmodule a) N, Cα, C’ (secondary 0.671 1.178 1.191 1.038 0.954 0.941 structure elements b) Heavy Atoms (core R1.945 2.840 2.732 2.455 2.015 2.077 module a) Heavy Atoms (secondary 1.163 1.933 1.649 1.521 1.379 1.354 structure elements b) a Core-R-modules are from the first identical amino (G1 in AlgE4R, G3 in AlgE6R1, G11 in AlgE6R2 and G11 in AlgE6R3 see also Fig. S1) acid until the C-terminal end of AlgE6R1 and AlgE6R2 or the amino acid that is in the same position when the 4 structures are aligned (T150 in AlgE4R and A162 in AlgE6R3). b The secondary structure elements are only the β-strands (Fig. 1 and 2)
R-modules in alginate epimerases Tab. 4: Dissociation constants and other thermodynamic data for the R-module from AlgE4 determined at 25C. Calculations from NMR data were done assuming a 1:1 (N = 1) complex. Nearly all the ITC data could be fitted to N = 1.M8 and M9 could not be fitted with one binding event. Data obtained by calculating two binding events are indicated by #. NMR
ITC
Ligand
Kd[µM]
N
Kd[µM]
ΔH [kJ/mol] -ΔST[kJ/mol]
M3
883 ± 98.1
Fixed to 1
323 ± 3.8
-33.8 ± 0.2
-13.9
-19.9 ± 1.4
M4
191 ± 27.2
Fixed to 1
26 ± 2.0
-45.4 ± 1.4
-19.3
-26.1 ± 2.4
M5
2.72 ± 1.91 Fixed to 1
3.75 ± 0.06 -41.9 ± 0.2
-10.9
-31.0 ± 1.1
M6
0.41 ± 0.30 Fixed to 1
1.03 ± 0.25 -46.8 ± 1.7
-12.6
-34.2 ± 2.1
M7
---
Fixed to 1
0.46 ±0.02
-45.0 ± 0.2
-8.8
-36.2 ± 0.9
M8 -1 #
---
0.499 ± 0.016 0.36 ± 0.02 -44.1 ± 3.1
-7.4
-36.7 ± 3.2
M8 - 2 # ---
0.465 ± 0.017 0.09 ± 0.01 -83.2 ± 2.2
-42.8
-40.4 ± 4.8
M9 - 1 # ---
0.418 ± 0.018 0.57 ± 0.03 -11.6 ± 6.3
24.0
-35.6 ± 6.7
M9 - 2 # ---
0.601 ± 0.02
0.15 ± 0.02 -91.8 ± 1.8
-52.9
-38.9 ± 5.6
MG4
156 ± 23.4
Fixed to 1
2040 ± 110 -47.8 ± 1.55
-32.4
-15.4 ± 3.6
MG5
---
Fixed to 1
4115 ± 239 -230 ± 12.8
-216.1
-13.9 ± 25.2
MG6
---
Fixed to 1
1204 ± 67
-98.5 ± 2.99
-81.9
-16.6 ± 8.7
MG7
314 ± 75.2
---
---
---
---
---
MG8
---
Fixed to 1
1330 ± 132 -210 ± 20.1
-652.1
-15.9 ± 27.9
ΔG [kJ/mol]
23
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Tab. 5: Distribution of M and G units obtained from endpoint epimerisation of poly-M. Monomer, diades, triades and average G-block length calculated based on 1H NMR spectra obtained from poly-M epimerised with AlgE46 or AlgE64. FG FM FGG FMG/GM FMM FMGG/GGM FMGM FGGG NG>1 AlgE4 0.46 0.54 0 0.46 0.08 0 0.46 0 0 AlgE46 0.41 0.59 0 0.41 0.17 0 0.41 0 0 AlgE6 0.62 0.38 0.52 0.11 0.272 0.03 0.09 0.52 12.0 AlgE64 0.82 0.18 0.75 0.08 0.10 0.06 0.07 0.69 16.0
Figure 1
----------GSD-GEPLVGGDTDDQLQGGSGADRLDGGAGDDILDGGAGRDRLSGGAGA ---------QGTDGNDVLIGSDVGEQISGGAGDDRLDGGAGDDLLDGGAGRDRLTGGLGA DPSAEAQPIVGSDLDDQLHGTLLGEEISGGGGADQLYGYGGGDLLDGGAGRDRLTGGEGA DPGVEGTPVVGSDLDDELHGTLGSEQILGGGGADQLYGYAGNDLLDGGAGRDKLSGGEGA *:* .: * * .::: **.* *:* * .*.*:********:*:** ** DTFVFSAREDSYRTDTAVFNDLILDFEASEDRIDLSALGFSGLGDGYGGTLLLKTNAEGT DTFRFALREDSHRSPLGTFSDLILDFDPSQDKIDVSALGFIGLGNGYAGTLAVSLSADGL DTFRFALREDSQRSAAGTFSDLILDFDPTQDKLDVSALGFTGLGNGYAGTLAVSVSDDGT DTFRFALREDSHRSPLGTFGDRILDFDPSQDRIDVSALGFSGLGNGYAGSLAVSVSDDGT *** *: **** *: ..*.* ****:.::*::*:***** ***:**.*:* :. . :* RTYLKSFEADAEGRRFEVALDGDHTGDLSAANVVFAATGTTT--ELEVLGDSGTQAGAIV RTYLKSYDADAQGRSFELALDGNHAATLSAGNIVFAAATPG------------------RTYLKSYETDAEGRSFEVSLQGNHAAALSADNILFATPVPV------------------RTYLKSYEADAQGLSFEVALEGDHAAALSADNIVFAATDAAAAGELGVIGASGQPDDPTV ******:::**:* **::*:*:*:. *** *::**:. .
AlgE4R AlgE6R1 AlgE6R2 AlgE6R3
AlgE4R AlgE6R1 AlgE6R2 AlgE6R3
AlgE4R AlgE6R1 AlgE6R2 AlgE6R3
167 152 161 180
109 111 120 120
49 51 60 60
Figure 2:
Figure 3
B.
D.
A.
C.
Figure 4
Figure 5
0 9.70
20
40
60
80
100
120
Relative height
9.60
9.55 Chemical Shift [ppm]
9.65
9.50
9.45
Figure 6
A. B.
Figure 7
Figure 8
D15
C.
A.
R51
R53
D26
R24
R42
R40
D85
K103
D94
E128
D130
K125
R121 D118
E137
R73
D74
R114
E117
R110 D130 E107
E126
R62
R124
D.
E17
D15
D28
E128
R64
K53
R51 R82
D85
E141
R121
K125
D109
K116
E138
R73
D132 R112 D76
R44
D24 R42
R26
B.
D119
D118
Protein Structure and Folding: Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii Edith Buchinger, Daniel H. Knudsen, Manja A. Behrens, Jan Skov Pedersen, Olav A. Aarstad, Anne Tøndervik, Svein Valla, Gudmund Skjåk-Braek, Reinhard Wimmer and Finn L. Aachmann
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Supplemental material: http://www.jbc.org/content/suppl/2014/09/29/M114.567008.DC1.html This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/early/2014/09/29/jbc.M114.567008.full.html#ref-list-1
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J. Biol. Chem. published online September 29, 2014
