S1 Understanding a mechanism of organic co-solvent inactivation in heme monooxygenase P450 BM-3 Jochen Kuper, † Tuck Seng Wong, ‡ Danilo Roccatano, ‡ Matthias Wilmanns, † and Ulrich Schwaneberg *,‡ † EMBL-Hamburg Outstation, DESY, Hamburg, Germany, and ‡ Jacobs University Bremen, School of Engineering and Science, Campus Ring 1, 28759 Bremen, Germany. Supporting Information 1 Materials and methods 1.1 General methods All chemicals used were of analytical reagent grade or higher quality and purchased from Sigma-Aldrich Chemie (Taufkirchen, Germany), Applichem (Darmstadt, Germany) and Carl Roth (Karlsruhe, Germany). A thermal cycler (Mastercycler gradient; Eppendorf, Hamburg, Germany) and thin- wall PCR tubes (Mµlti-Ultra tubes; 0.2 mL; Carl Roth, Germany) were used in all PCRs. The PCR volume was always 50 µL; larger volumes were prepared in multiple 50 µL PCRs. The amount of DNA was quantified using a NanoDrop photometer (NanoDrop Technologies, Wilmington, Delaware, USA). All PCR purification and gel extraction were performed using QIAquick PCR purification kit (Qiagen, Düren, Germany) and QIAquick gel extraction kit (Qiagen) respectively. Enzymes were purchased from New England Biolabs (Frankfurt, Germany), unless otherwise stated. 1.2 Cloning of P450 BM-3 heme domain (BMP) into pETM-11 vector Gene segment encoding P450 BM-3 heme domain (BMP, Thr1-Leu455) was amplified from pCWori vector 1 harboring full-length P450 BM-3 gene (encoding both heme and reductase domains). For the PCR (95 o C for 30 s, 1 cycle; 95 o C for 30 s/55 o C for 1 min/68 o C for 2 min, 30 cycles; 68 o C for 10 min, 1 cycle), 2.5 U Pfu Turbo DNA polymerase (Stratagene Europe, Amsterdam Zuidoost, Netherlands), 0.2 mM dNTP mix (New England Biolabs), 20 pmol of each primer (5’- [Phos]CATGACAATTAAAGAAATGCCTC-3’ and 5’- CTGGAATTCTTAAAGCGGAATTTTTTTCGATTT-3’) and 100 ng template (pCWori vector harboring P450 BM-3 gene) were used. The PCR product was subsequently ligated into pETM-11 vector using NcoI and EcoRI restriction sites, followed by transformation into E. coli XL2 Blue (Stratagene) using TSS method. 2 1.3 Expression of P450 BM-3 heme domain (BMP) in shaking flask pETM-11 vector harboring P450 BM-3 heme domain gene was freshly transformed into E. coli BL21 (DE3). Single colony was picked to prepare overnight culture in 5 mL LB kan media. Three hundred mL TB kan media, supplemented with 300 µL trace element (0.5 g CaCl 2 ·2H 2 O, 0.18 g ZnSO 4 ·7H 2 O, 0.10 g MnSO 4 ·H 2 O, 20.1 g Na 2 - EDTA, 16.7 g FeCl 3 ·6H 2 O, 0.16 g CuSO 4 ·5H 2 O, 0.18 g CoCl 2 ·6H 2 O, add 1 L H 2 O and autoclave), were inoculated with 1.5 mL overnight culture. The culture was incubated (37 o C, 250 rpm, ~3 h; Multitron II; Infors GmbH, Einsbach, Germany) and * To whom correspondence addressed: u.schwaneberg@iu-bremen.de