Supporting Information for:
Directing
positional
specificity
in
enzymatic
synthesis
of
bioactive
1-
phosphatidylinositol by protein engineering of a phospholipase D
Jasmina Damnjanović, Chisato Kuroiwa, Hidetoshi Tanaka, Ken Ishida, Hideo Nakano
and Yugo Iwasaki*
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences,
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Running title: Engineering of 1-PI specific phospholipase D
Keywords: phospholipase D, 1-phosphatidylinositol, positional specificity
Figure S1. Outline of the overlapping PCR used to introduce single residue mutations.
A 1.8 kbp fragment was amplified using primers CATR1 and one of the four reverse primers
(NYR-186X-Rv, NYR-188X-Rv, NYR-189X-Rv or NYR-190X-Rv listed in Table SI) with
nyr as a template. Another 1.0 kbp fragment was amplified using one of the specific forward
primers listed in Table SI and T7T with nyr as a template. The two fragments were joined by
overlapping PCR using primers, CATR1 and T7T to afford the full length PLD gene of 2.8
kbp containing single residue mutation.
Figure S2. Analysis of the protein-ligand interactions in crystal structure of Streptomyces
antibioticus PLD (PDB: 2ZE9). Ligand: 1,2-diheptanoyl-sn-glycero-3-phosphate.
3-PI
(%)
1-PI
(%)
Figure S3. 1-PI/3-PI ratio produced by NYR-186X/189X double mutants, as determined by
LC-MS analysis. As a reference, parent NYR produces 76% of 1-PI and 24% of 3-PI.
Figure S4. SDS-PAGE of the purified enzyme fractions (10% gel): lane M- molecular
weight marker (Takara, Japan) followed by lanes of the purified NYR and its variants.
Figure S5. Presentation of the polar interactions, including hydrogen bonds, (yellow dots)
formed between myo-inositol and residues of the active site and acceptor-binding site after
docking of inositol to the phosphatidyl-bound model structures of NYR and GYR. Ptd
denotes phosphatidyl moiety while myo-Ins denotes myo-inositol. Backbone of inositol and
phosphatidyl moiety is colored light blue while, that of the residues is colored green. Oxygen
atoms are colored red, hydrogen atoms white and nitrogen atoms dark blue. Phosphorus is
presented in orange color.
Figure S6. Analysis of hydrogen bond network formed by residue 188 in model structures of
NYR (K188), NYR-188W, NYR-188E and NYR-188N. Hydrogen bonds are colored yellow.
Red, blue and magenta boxes indicate main chain-main chain, side chain-side chain and main
chain-side chain interactions, respectively.
Table SI. List of the primers used in this study.
Primer Name
Sequence (5’-3’)
a) Generation of G186X variants
NYR-G186A
ACGGGCGGGATCAACGCCAACAAGGACGACTA
NYR-G186C
ACGGGCGGGATCAACTGCAACAAGGACGACTA
NYR-G186D
ACGGGCGGGATCAACGACAACAAGGACGACTA
NYR-G186E
ACGGGCGGGATCAACGAGAACAAGGACGACTA
NYR-G186F
ACGGGCGGGATCAACTTCAACAAGGACGACTA
NYR-G186K
ACGGGCGGGATCAACAAGAACAAGGACGACTA
NYR-G186H
ACGGGCGGGATCAACCACAACAAGGACGACTA
NYR-G186I
ACGGGCGGGATCAACATCAACAAGGACGACTA
NYR-G186L
ACGGGCGGGATCAACCTGAACAAGGACGACTA
NYR-G186M
ACGGGCGGGATCAACATGAACAAGGACGACTA
NYR-G186N
ACGGGCGGGATCAACAACAACAAGGACGACTA
NYR-G186P
ACGGGCGGGATCAACCCGAACAAGGACGACTA
NYR-G186Q
ACGGGCGGGATCAACCAGAACAAGGACGACTA
NYR-G186R
ACGGGCGGGATCAACCGCAACAAGGACGACTA
NYR-G186S
ACGGGCGGGATCAACTCGAACAAGGACGACTA
NYR-G186T
ACGGGCGGGATCAACACCAACAAGGACGACTA
NYR-G186V
ACGGGCGGGATCAACGTCAACAAGGACGACTA
NYR-G186W
ACGGGCGGGATCAACTGGAACAAGGACGACTA
NYR-G186Y
ACGGGCGGGATCAACTACAACAAGGACGACTA
NYR-G186X-Rv
GTTGATCCCGCCCGCCCGT
b) Generation of K188X variants
NYR-K188A
GGGATCAACGGCAACGCGGACGACTACCTCGAC
NYR-K188C
GGGATCAACGGCAACTGCGACGACTACCTCGAC
NYR-K188D
GGGATCAACGGCAACGACGACGACTACCTCGAC
NYR-K188E
GGGATCAACGGCAACGAGGACGACTACCTCGAC
NYR-K188F
GGGATCAACGGCAACTTCGACGACTACCTCGAC
NYR-K188G
GGGATCAACGGCAACGGGGACGACTACCTCGAC
NYR-K188H
GGGATCAACGGCAACCACGACGACTACCTCGAC
NYR-K188I
GGGATCAACGGCAACATCGACGACTACCTCGAC
NYR-K188L
GGGATCAACGGCAACCTGGACGACTACCTCGAC
NYR-K188M
GGGATCAACGGCAACATGGACGACTACCTCGAC
NYR-K188N
GGGATCAACGGCAACAACGACGACTACCTCGAC
NYR-K188P
GGGATCAACGGCAACCCGGACGACTACCTCGAC
NYR-K188Q
GGGATCAACGGCAACCAGGACGACTACCTCGAC
NYR-K188R
GGGATCAACGGCAACCGCGACGACTACCTCGAC
NYR-K188S
GGGATCAACGGCAACTCGGACGACTACCTCGAC
NYR-K188T
GGGATCAACGGCAACACGGACGACTACCTCGAC
NYR-K188V
GGGATCAACGGCAACGTGGACGACTACCTCGAC
NYR-K188W
GGGATCAACGGCAACTGGGACGACTACCTCGAC
NYR-K188Y
GGGATCAACGGCAACTACGACGACTACCTCGAC
NYR-K188X-Rv
GTTGCCGTTGATCCCGCCCG
c) Generation of D189X variants
NYR-D189A
ATCAACGGCAACAAGGCCGACTACCTCGACACC
NYR-D189C
ATCAACGGCAACAAGTGCGACTACCTCGACACC
NYR-D189E
ATCAACGGCAACAAGGAGGACTACCTCGACACC
NYR-D189F
ATCAACGGCAACAAGTTCGACTACCTCGACACC
NYR-D189G
ATCAACGGCAACAAGGGCGACTACCTCGACACC
NYR-D189H
ATCAACGGCAACAAGCACGACTACCTCGACACC
NYR-D189I
ATCAACGGCAACAAGATCGACTACCTCGACACC
NYR-D189K
ATCAACGGCAACAAGAAGGACTACCTCGACACC
NYR-D189L
ATCAACGGCAACAAGCTGGACTACCTCGACACC
NYR-D189M
ATCAACGGCAACAAGATGGACTACCTCGACACC
NYR-D189N
ATCAACGGCAACAAGAACGACTACCTCGACACC
NYR-D189P
ATCAACGGCAACAAGCCGGACTACCTCGACACC
NYR-D189Q
ATCAACGGCAACAAGCAGGACTACCTCGACACC
NYR-D189R
ATCAACGGCAACAAGCGCGACTACCTCGACACC
NYR-D189S
ATCAACGGCAACAAGTCGGACTACCTCGACACC
NYR-D189T
ATCAACGGCAACAAGACCGACTACCTCGACACC
NYR-D189V
ATCAACGGCAACAAGGTCGACTACCTCGACACC
NYR-D189W
ATCAACGGCAACAAGTGGGACTACCTCGACACC
NYR-D189Y
ATCAACGGCAACAAGTACGACTACCTCGACACC
NYR-D189X-Rv
CTTGTTGCCGTTGATCCCGC
d) Generation of D190X variants
NYR-D190A
ACGGCAACAAGGACGCGTACCTCGACACCG
NYR-D190C
ACGGCAACAAGGACTGCTACCTCGACACCG
NYR-D190E
ACGGCAACAAGGACGAGTACCTCGACACCG
NYR-D190F
ACGGCAACAAGGACTTCTACCTCGACACCG
NYR-D190G
ACGGCAACAAGGACGGCTACCTCGACACCG
NYR-D190H
ACGGCAACAAGGACCACTACCTCGACACCG
NYR-D190I
ACGGCAACAAGGACATCTACCTCGACACCG
NYR-D190K
ACGGCAACAAGGACAAGTACCTCGACACCG
NYR-D190L
ACGGCAACAAGGACCTCTACCTCGACACCG
NYR-D190M
ACGGCAACAAGGACATGTACCTCGACACCG
NYR-D190N
ACGGCAACAAGGACAACTACCTCGACACCG
NYR-D190P
ACGGCAACAAGGACCCGTACCTCGACACCG
NYR-D190Q
ACGGCAACAAGGACCAGTACCTCGACACCG
NYR-D190R
ACGGCAACAAGGACCGCTACCTCGACACCG
NYR-D190S
ACGGCAACAAGGACTCGTACCTCGACACCG
NYR-D190T
ACGGCAACAAGGACACGTACCTCGACACCG
NYR-D190V
ACGGCAACAAGGACGTCTACCTCGACACCG
NYR-D190W
ACGGCAACAAGGACTGGTACCTCGACACCG
NYR-D190Y
ACGGCAACAAGGACTACTACCTCGACACCG
NYR-D190X-Rv
GTCCTTGTTGCCGTTGATCCC
e) Sequencing and common primers
pld-seq1
TGGCTGCTGCACACCCCCGGCT
pld-seq2
ATCTACCACCTCAACGTGGTGC
pld-seq3
ACCCGGTGTCGGACGTGGACATGG
pld-seq4
ACCCCTCCTCGGGATACCACCCGGA
pld-seq5
CGCCGGGTCAAGGTCCGCATCG
T7P
TAATACGACTCACTATAGGG
T7T
GCTAGTTATTGGTCAGCGG
CATR1
TAGCAGATCTGAGCTCACTAGTGGATCCTCGAATTTCTGCCATTCAT