Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Applied Microbiology and Biotechnology
Supporting Information for
Iterative Key-residues interrogation of a phytase harboring
thermostability increasing substitutions identified in directed
evolution
Amol V. Shivangea,b,c, Danilo Roccatanob and Ulrich Schwaneberga,b *
a
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany
b
School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759
Bremen, Germany
c
Present Address: Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, California 91125, United States
Running Title: Phytase: structure function relationships
*Corresponding author: Prof. Dr. Ulrich Schwaneberg
RWTH Aachen University
Lehrstuhl für Biotechnologie
Worringer Weg 1
52056 Aachen, Germany
Tel.: +49-241-80-24170
Fax: +49-241-80 22387
E-Mail: u.schwaneberg@biotec.rwth-aachen.de
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Homology modeling of Ympytase
Template selection
The
amino
acid
sequence
of
Ymphytase
was
subjected
to
NCBI-BLAST
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to identify homologous structure for Ymphytase by
searching the structural database of protein sequences in the Protein Data Bank (PDB) (Berman et
al. 2000). A template, E. coli phytase (1DKM) was selected based on highest percentage sequence
identity (45%) and highest resolution of the crystal structure (Electronic supplementary material,
Table S3 and Fig. S9).
Sequence alignment and model building
Sequence alignment of the query sequence with respective template was performed using
dynamic programming alignment algorithm implemented in MODELLER (Sali 1996). This
method provides an alignment slightly different from standard sequence-sequence alignment
methods since it takes into account structural information from the template when constructing an
alignment. This is done by using a variable gap penalty function that places gaps in solvent exposed
regions, curved regions, and outside secondary structural elements (Supporting Information Fig.
S9). Subsequently, the alignment was used by the same program to model the Ymphytase protein
using 1DKM as a template structure.
Validation of homology models
The accuracy and validity of the generated homology model was analyzed with PROCHECK
server implemented in SWISS-MODEL server (http://swissmodel.expasy.org) and 3D-profile
programs (Bowie et al. 1991) (http://nihserver.mbi.ucla.edu/Verify_3D/). The latter calculates 3D
to 1D compatibility score and performs Eisenberg analysis for depiction of correctly folded and
misfolded regions in the protein structure.
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S1 PROCHECK-Ramachandran plot for the homology model of YmPhytase
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S2 3D-profile window plots for Ymphytase homology model. The vertical axis shows the
average 3D-1D score for the residue and horizontal line indicate residue number in the model.
The residues with average positive score in 3D-1D slide window are reasonably folded.
Fig. S3 Radius of gyration of the backbone atoms (Cα, N, C) of Ymphytase throughout the
simulation of wild-type (a; Wt) and variant M6 (b)
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S4 Structural topology of Ymphytase model obtained from molecular dynamics simulation
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S5 Hydrogen bonding network of Ymphytase wild-type deciphered by MD simulation.
Amino acid residue G187 showed no hydrogen bonds with loop-G6 (a), T77 did not show any
hydrogen bonds with D122 (b), K289 showed 2 – 3 hydrogen bonds with helix-L (c) but the
frequency of second hydrogen bond was less compare to variant M6 and negligible hydrogen
bonds with loop-CD (d)
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S6 Salt bridge analysis of T77K with D122 showing the minimum distance between residues
K77 and D122 throughout the simulation time. The average distance between K77 and D122 was
0.37 nm showing a salt bridge interaction (centroids of the side-chain charged group atoms in the
residues lie within 0.4 nm of each other)
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S7 A tetrameric model of Ymphytase constructed by superimposing monomers to biological
assembly of Debaryomyces castellii phytase (PDB ID: 2GFI). Substitutions in Ymphytase variant
M6 are shown in orange colored space filled model. Highly flexible loops observed in MD
simulation (L92-T102, N247-P252, and Q304-Q317) are shown in red color. Residues involved in
inter-monomer interaction calculated using PIC server (http://crick.mbu.iisc.ernet.in/~PIC/) are
depicted in purple (hydrophobic interactions), yellow (ionic interactions), and blue (hydrogen
bonding interactions) color
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Fig. S8 Hydrogen bonding network of Ymphytase residue Q154 in wild-type (a) and
substitution Q154H in variant M6 over the time of MD simulation. Hydrogen bonds were
calculated between residue 154 and the protein molecule
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Table S1 Sequence of the primers used for site-directed and site saturation mutagenesis
Primer name
Sequence
Ymph_D52N_AS_F
CAGAGTTAATGAATAATGTCACACCGGATAAG
Ymph_T77K_AS_F
GGTGCGCAATTAGTGAAACTGATGGGCGGCTTC
Ymph_K139E_AS_F
CATTATCAGGCTGATTTGGAGAAGGTTGATCCACTG
Ymph_G187S_AS_F
CCATTTGCCCAGATGAGCGAGATTCTCAATTTC
Ymph_V298M_AS_F
GTTGTTGCAACAGATTATGACGGCGCTAGTGCTTC
YmG81E-F_AS
GTGACACTGATGGGCGAATTCTATGGTGATTATTTC
YmQ154H-F_AS
CCGGTGTGTGTCATCTAGATTCGACAC
YmL239V-F_AS
CACTCTCATCCACAGTGGGTGAAATCTTCTTG
YmK289E-F_AS
CTTATATCGCCCGTCATGAGGGAACTCCGTTGTTG
YmK289X-F_AS
CTTATATCGCCCGTCATNNKGGAACTCCGTTGTTG
Table S2 Classification and mutational pattern of the variants identified in the 2nd round of
SeSaM library screening
Group
Variant
Template SM2P3E4
Group I SM3P1F3
SM3P1B4
Group II
SM3P3D20
SM3P1D5
SM3P1F10
SM3P1F1
Group
SM3P1A5
III
SM3P1B2
SM3P1A6
SM3P1C1
SM3P1A3
Group
IV
SM3P1C12
Substitutions
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M, Q154H, L239V
R2H, D52N, T77K, K139E, G187S, V298M, K289E
R2H, D52N, T77K, K139E, G187S, V298M, G81E
R2H, D52N, T77K, K139E, G187S, V298M, V20L, G81D, K422R
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M, V24M
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M
R2H, D52N, T77K, K139E, G187S, V298M, S260T
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
Table S3 Available templates for homology modeling of YmPhytase (*selected template)
Template
PDBID
%
Identity
Resolution
(Å)
Mutation
(s)
Ligand
1DKL
45
2.30
No
No
1DKM*
45
2.25
A116T
No
1DKP
44
2.28
A116T,
H17A
Phytate
1NT4
33
2.40
H18A
Glucose-1Phosphate
Fig. S9 Structure-sequence alignment of template and query sequence based on dynamic
programming algorithm of MODELLER for YmPhytase.
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
120
Relative activity (%)
100
M6
Wt
M3
80
60
40
20
0
2
3
4
5
6
pH
7
8
9
10
Fig. S10 Activity-pH profile of the Ymphytase wildtype and variant M3 and M6.
Native polyacrylamide gel electrophoresis (PAGE) of wildtype Ymphytase and variant M3
and M6
Native polyacrylamide gel (10%) was prepared by omitting SDS from the routinely used
polyacrylamide gel electrophoresis. NativeMark Unstained Protein Standard (Invitrogen) was used
as a molecular marker to estimate the quaternary form of Ymphytase variants. Purified protein of
Ymphytase variants [7 μL of protein and 3 μL of loading dye (tris 62.5 mM pH 6.8, glycerol 30%,
bromophenol blue 0.01%)] were loaded into each lane of the native gel. The electrophoresis was
performed using tris–glycine buffer (pH 8.3) and electrophoresis chamber was placed in an icewater bath during the run. Protein bands were stained with Coomassie brilliant blue solution.
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Supporting Information
Shivange et al.
Structure function relationships of Yersinia mollaretii phytase
References
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE
(2000) The Protein Data Bank. Nucleic Acids Res 28(1):235-42 doi:10.1093/nar/28.1.235
Bowie JU, Luthy R, Eisenberg D (1991) A method to identify protein sequences that fold into a
known three-dimensional structure. Science 253(5016):164-70
Sali A (1996) Comparative protein modeling by satisfaction of spatial restraints.
Immunotechnology 2:279-80
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Supporting Information