Fig. S1
A622
A622L
1
1
MVVSEKSKILIIGGTGYIGKYLVETSAKSGHPTFALIRESTLKNPEKSKLIDTFKSYGVT
..................................V.......V.................
60
60
A622
A622L
61
61
LLFGDISNQESLLKAIKQVDVVISTVGGQQFTDQVNIIKAIKEAGNIKRFLPSEFGFDVD 120
...............................A............................ 120
A622
A622L
121 HARAIEPAASLFALKVRIRRMIEAEGIPYTYVICNWFADFFLPNLGQLEAKTPPRDKVVI 180
121 ..H.............K........................................... 180
A622
A622L
181 FGDGNPKAIYVKEEDIATYTIEAVDDPRTLNKTLHMRPPANILSFNEIVSLWEDKIGKTL 240
181 ....................MK...............................E...... 240
A622
A622L
241 EKLYLSEEDILQIVQEGPLPLRTNLAICHSVFVNGDSANFEVQPPTGVEATELYPKVKYT 300
241 ...........H......M...V..................I..S............... 300
A622
A622L
301 TVDEFYNKFV 311
301 ....Y..... 311 (Identity=95.5%, Similarity=98.4%)
Fig. S2
189
135
238
204
231
220
103
(73.0%)
(85.9%)
(68.4%)
236
211
136
A622
A622L
189
(98.4%)
135
(98.5%)
238
(97.1%)
167
(nt)
135
(43.3%)
245
204
(96.6%)
167
(93.4%)
Fig. S3
Red: A622
Blue: ObEGS1+NADPH
Green: PtPCBER
Supplementary Figures
Figure S1. Alignment of the amino acid sequences of A622 and A622L. Dots indicate the A622L residues
that are identical to those of the A622 sequence.
Figure S2. Diagram of the A622 and A622L genes. Filled boxes indicate coding exons, whereas the lines
show introns. The numbers indicate the lengths of the exons and introns in nucleotides. The sequence
identity values between corresponding gene segments are indicated in parentheses. The A622L genomic
sequence was deposited in GenBank under the accession number AB445396.
Figure S3. Superposition of the polypeptide-chain backbones of A622, ObEGS1 (PDB entry no. 2R6J),
and PtPCBER (PDB entry no. 1qyc). The color coding is shown in the inset. The NADPH cofactor in
ObEGS1 is shown in the ball mode. The structural modeling of A622 protein and the comparison with
other PIP-family proteins were carried out using MODELLER 8v2 (University of California San Francisco,
San Francisco, CA) and PyMol version 0.99rc6 (DeLano Scientific LLC, Palo Alto, CA) software,
respectively.
Supplementary Materials and Methods
Determination of the A622L genomic and cDNA sequences
Since the A622L genomic sequence in the N. tabacum genome database
(http://www.tobaccogenome.org/) was incomplete, genomic PCR was carried out with the primers 5’CCTCCACCTTAACCCGAAGC and 5’-TGCAGATTGATGTCGACAAC, using 10 ng of N. tabacum
genomic DNA and TaKaRa Ex Taq DNA polymerase (TaKaRa Bio) under the following conditions: 30
cycles of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1.5 min. The PCR product was sequenced
directly by using 5’-TCAGAGAAAGCACACTCGT and 5'-GCATATGGCCAAATTGACT.
To determine the cDNA sequence of A622L, RT-PCR was carried out with 5’CCTCCACCTTAACCCGAAGC and 5’-TGCAGATTGATGTCGACAAC, using 1 ng of N. tabacum root
tissue cDNA and TaKaRa Ex Taq DNA polymerase (TaKaRa Bio) under the following conditions: 30 cycles
of 94 °C for 30 sec, 55 °C for 30 sec, and 72 °C for 1 min. The PCR product was cloned into pGEM-T
easy vector (Promega, Madison, WI), and sequenced using 5’-TCAGAGAAAGCACACTCGT and 5'GCATATGGCCAAATTGACT.