Supporting Information. Copyright Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2008

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1 Supporting Information Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 9 Weinheim, 008

2 Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 9 Weinheim, 008 Supporting Information for Streptomyces Phospholipase D Mutants With Altered Substrate Specificity Capable of Phosphatidylinositol Synthesis Atsushi Masayama, Tetsuya Takahashi, Kaori Tsukada, Seigo Nishikawa, Rie Takahashi, Masaatsu Adachi, Kazushi Koga, Atsuo Suzuki, Takashi amane, Hideo Nakano, and ugo Iwasaki*

3 ibrary construction Col El ori Amp r ppeb-pd-ks-ii-cat P-F T7-lac Promoter -F 87 NNS 9 NNS -F 8 NNS Xho I Spe I cat PCR rbs signal pld -R PCR -R P-R PCR NNS NNS NNS verlapping PCR NNS NNS NNS verlapping PCR Xho I Spe I cat NNS NNS pld NNS Col El ori Col El ori Cut with Spe I & Xho I petkms-term. igation Spe I T7-term Sal I Introduce into E. coli DH a Figure S. Schematic representation of PCR-assisted mutagenesis.

4 PI-synthesizing activity of mutant PDs PI W F R F T C F G F V W M S E S S R F M M F R V V W V A G W D E W R S R R F R R H N M A V A A F 87th 9st 8th PA PI PC Figure S. TC analysis of PI formation by mutant PDs. Positions of the phospholipids are indicated. The mutant PDs are indicated by using triplets denoting the amino acids at positions 87, 9, and 8. ane PI denotes the soybean PI as the authentic standard. Table S. DNA sequence and amino acid sequence of positive mutants. Mutant Position Frequency of appearance WT TGG(W) TAC() TAC() - TTC (F) AGG (R) TAC () 9 TTC (F) ACG (T) TGC (C) TTC (F) GGC (G) TAC () TTC (F) GTG (V) CTC () TGG (W) TAC () ATG (M) TCG (S) TAC () GAG (E) 7 TCC (S) AGG (R) ATG (M) 8 TCC (S) TTC (F) ATG (M) 9 TAC () TTC (F) TAC () 0 TAC () TTG () AGG (R) TAC () TTG () TAC () GTG (V) TAC () TAC () GTG (V) TAC () TGG (W) GTC (V) TAC () GAC (E) GCG (A) TGG (W) TGG (W) GGC (G) GAC (D) CTG () 7 CGG (R) TAC () AGC (S) 8 CGG (R) TAC () TAC ()

5 9 CGG (R) TAC () TTC (F) 0 AGG (R) AGG (R) TAC () CAC (H) ATG (M) GTC (V) AAC (N) GCG (A) TAC () TTG () GCG (A) TAC () TTG () GCG (A) TTC (F) TTG () TTG () TAC () The # mutant (FR) was sequenced in full length and confirmed to have no other mutation. The other mutants were not sequenced in full length, and they might contain unexpected mutations. NMR analysis of enzymatically synthesized PI isomers. Ins P * Glycerol ' ' ' ' ' ' 7' 8' 9' 0' ' ' ' ' ' ' 7' 8' le and P Ins * Glycerol ' ' ' ' ' ' 7' 9' 0' ' ' ' 8' ' ' ' 7' 8' le Figure S. Structure of compound (Figure A, lane ). H NMR (00 MHz, CDCl /CD D = /, 0 K): δ 0.80 (H, t, J =.8 Hz, le-h-8,8'),..8 (0H, m, le),.9. (H, m, le-h-,'), (8H, m, le-h-8,8',,'),. (H, dt, J = 7. Hz, J =. Hz, le-h-,'),.8 (H, m, Ins-H-),. (H, m, Ins-H- or Ins-H-),.7 (H, m, Ins-H- or Ins-H-),.7 (H, m, Ins-H- or Ins-H-),.8 (H, m, Ins-H- or Ins-H-),.9.00 (H, m, glycerol-h-,'),.08. (H, m, Ins-H-, glycerol-h-),. (H, m, glycerol-h-'),.8 (H, m, glycerol-h-),..8 (H, m, le-h-9,9',0,0').

6 Ins P * Glycerol ' ' ' ' ' ' 7' 9' 0' ' ' ' 8' ' ' ' 7' 8' le and P Ins * ' Glycerol ' ' ' ' 7' ' 8' 9' 0' ' ' ' 8' ' ' ' 7' le Figure S. Structure of compound (Figure A, lane ). H NMR (00 MHz, CDCl /CD D = /, 0 K): δ 0.8 (H, t, J =.0 Hz, le-h-8,8'),..8 (0H, m, le),.8.7 (H, m, le-h-,'), (8H, m, le-h-8,8',,'),. (H, dt, J = 7.8 Hz, J =. Hz, le-h-,'),.0 (H, m, Ins-H- or Ins-H-),.7 (H, m, Ins-H-),.8 (H, m, Ins-H- or Ins-H-),.8 (H, m, Ins-H- or Ins-H-),.9.00 (H, m, Ins-H- or Ins-H-, glycerol-h-,'),. (H, m, glycerol-h-),. (H, m, Ins-H-),. (H, br-d, J = 0. Hz, glycerol-h- '),.0 (H, m, glycerol-h-),..9 (H, m, le-h-9,9',0,0'). Ac Ac Ac Ac Ins ' Ac P Glycerol * ' ' ' ' 7' ' 8' 9' 0' ' ' ' ' ' ' 7' 8' le and Ac ' Ac Ac P Ac Ac Ins Glycerol * ' ' ' ' ' 7' 8' 9' 0' ' ' ' ' ' ' 7' 8' le Figure S. Structure of compound (Figure A, lane, acetylated). H NMR (00 MHz, CDCl ): δ 0.88 (H, t, J =. Hz, le-h-8,8'),.. (0H, m, le),.. (H, m, le-h-,'),.9.0 (H, m le-h-8,8',,', Ac x ),.0 (H, le-h-,'),.9 (H, m, glycerol-h-,'),.7 (H, m, glycerol-h-),.8 (H, br-d, J =. Hz, glycerol-h-'),. (H, m, Ins-H- or Ins-H-),. (H, m, Ins-H- or Ins-H-),..9 (7H, m, Ins-H-, or Ins-H-,, glycerol-h-, le-h-9,9',0,0'),.7 (H, m, Ins-H- or Ins-H-),. (H, br s, Ins-H-).

7 Figure S. H NMR of compound. Figure S7. H,H CS of compound.

8 Figure S8. H NMR of compound. Figure S9. H,H CS of compound. 7

9 Figure S0. H NMR of compound. Figure S. H,H CS of compound. 8

10 Figure S. Analysis of the transphosphatidylated product of,,-cyclohexanetriol. The enzymatic product in chloroform : methanol = : (lane ) was treated with an equal volume of 0% aqueous NaI solution at room temperature for 0 min with mixing. The lipid layer was recovered and analyzed by TC (lane ). Complete disappearance of PX was observed. Development of HPC method for PI isomer separation Preparation of standard PI isomers: Standard PI isomers were synthesized as shown in Figure S. Enantiomerically pure,,,-protected inositol (D-) and,,,-protected inositol (-) and racemic,,,(,,,)-protected inositols (rac-) were prepared as described. [9a,b],,,,-Protected inositol () was prepared as described. [9c,d] These protected myo-inositols were coupled with dioleoyl-pa in the presence of,,-triisopropylbenzenesulphonylchloride (TPS-Cl) according to [9e], followed by acid-mediated hydrolysis to afford the target compounds. The compound D- was used to synthesize a mixture containing -PI and -PI. The compound - was used for a mixture containing -PI and -PI, the compound rac- was used for a mixture of -PI, -PI and -PI, and the compound was used for -PI. These synthesized products were used for peak identification without isolating each isomer. 9

11 a b H -PI -PI a b H -PI -PI rac- a H b Me Me a Me Me c -PI Figure S. Preparation of PI isomers. a) DPA, TPS-Cl, pyridine, RT, h; b) CHCl /Me/ H /TFA = :::, RT, h. c) CHCl /Me/H /TFA =.7:.::, room temp., h. Peak identification: Figure SA shows the chromatogram of the mixture of -PI and -PI when detected by C-MS at m/z 8. [M-H]. Two peaks were detected at RT of.9 min and 7.7 min. Since this mixture contained -PI and -PI, one of the peaks was -PI and the other was -PI. The H NMR analysis of the FR-mediated reaction 0

12 products revealed that the faster eluting peak was either -PI and/or -PI and the slower eluting peak was -PI and/or -PI. Therefore, the peak at.9 min was -PI, and the one at 7.7 min was -PI. Figure SB is the chromatogram of the mixture of -PI and -PI. Similarly, the faster eluting peak was -PI and the other was -PI. From Figures SA and B, we realized that the separations of -PI from -PI and -PI from -PI were impossible due to structural symmetry of the myo-inositol moiety. Figure SC shows the separation of the mixture of -PI, -PI, and -PI. Since the faster eluting peak (.9 min) is that of co-eluting -PI and -PI, the other peak (. min) was assigned as -PI. Figure SD shows the separation of -PI, showing a single peak at.9 min. Figure SE shows the separation of the six isomers. Four peaks were base-line separated with the elution order of ()-PI, -PI, -PI, and ()-PI. A -PI -PI TIC B m/z=8. -PI -PI TIC C m/z=8. ()-PI -PI TIC D m/z=8. -PI TIC E m/z=8. ()-PI -PI -PI ()-PI 0 TIC m/z= Retention time (min) Figure S. Separation of authentic PI isomers by HPC. Separation of chemically syn- thesized mixtures of - and -PI (A); - and -PI (B); -, -, and -PI (C); -PI (D); and all six isomers (E) are shown.

Supplementary Appendix

Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Sherman SI, Wirth LJ, Droz J-P, et al. Motesanib diphosphate