Supplemental Information (SI)

Side chain removal from corticosteroids by unspecific peroxygenase

René Ullrich1#, Marzena Poraj-Kobielska1#, Steffi Scholze1, Claire Halbout2, Martin Sandvoss2, Marek J. Pecyna1,3, Katrin Scheibner5 and Martin Hofrichter1*

1 TU Dresden, International Institute Zittau, Department of Bio- and Environmental Sciences, Markt 23, 02763 Zittau, Germany

2 Sanofi-Aventis Deutschland GmbH, R&D, Integrated Drug Discovery IDD, Isotope Chemistry & Metabolite Synthesis ICMS, G876, 65926 Frankfurt/Hoechst, Germany

3 University of Applied Sciences Zittau-Görlitz, Faculty of Natural and Environmental Sciences, Theodor-Körner- Allee 16, 02763 Zittau, Germany

4 BTU Cottbus-Senftenberg, Faculty of Environment and Natural Sciences, Universitätsplatz 1, 01968, Senftenberg, Germany

* Corresponding author (e-mail address: [email protected])

# These authors contributed equally to this work

Marasmius wettsteinii and its unspecific peroxygenase (MweUPO)

The genus contains about 500 species of , most of which develop tiny inconspicuous fruiting bodies () in leaf-litter or on coarse woody debris. M. wettsteinii is a member of the subsection Marasmius and related to M. bulliardii (Quél.) and M. rotula. Antonin and Noordeloos described meticulously the differences of the basidiomata (M. wettsteinii ‘has, when fresh a white to cream colored that becomes brownish or beige on drying’) and the ecology. Unlike the other two taxa that prefer broad-leaved litter and hard-wood twigs, M. wettsteinii is typically dwelling needles of coniferous trees [1].

Our isolated strain (DSM 106021) produced appreciable peroxygenase (UPO) activities with a maximum of 2,500 U L-1 in an N- and C-rich complex medium [2], which represents an about 15-times lower level compared to M. rotula but is in the same range as the activities observed for the UPO- model Agrocybe aegerita [3] (Fig. S1). The isolation of the major isoenzyme of MweUPO by FPLC was successful and easier to achieve compared to the UPO of M. rotula (MroUPO) [2]. Thus, homogeneous MweUPO with 26% yield was obtained after four FPLC steps (Table S1), whereas for the purification of MroUPO, five steps were required (with an about 40-times lower yield) [2]. Determined molecular masses of MweUPO (and MroUPO) were dependent on the analytical method. Using a non- denaturing (native) method such as HP-SEC, the analysis resulted in 62 kDa (58 kDa for MroUPO); in contrast, denaturing SDS-PAGE gave about half the molecular mass for both enzymes (32.5 kDa and 32 kDa, respectively) (Fig. S2 and S3). This finding strongly indicates that ‘short UPOs’ as those of the genus Marasmius tend to form dimers under physiological conditions, which are linked by disulfide bridges originating from coupled cysteine residues [4].

Table S1: Purification of the major peroxygenase isoform of Marasmius wettsteinii (MweUPO)

Total Protein Specific Purifi- Yield Step activity amount activity cation (U) (mg) (U mg-1) (fold) (%) Ultrafiltration (cut off 10 kDa) 3,560 2,264 1.6 - 100 Q-Sepharose™ (26x200) 2,398 1,426 1.7 1 67 Mono Q™ (10x100) 2,134 595 3.6 2 60 Superdex™ 200 (26x600) 1,734 120 14.4 9 49 Mono Q™ (5x50) 917 24 37.5 24 26

NMR analyses

Table S2: 1H- and 13C data of P I in DMSO-d6

Pos. δC [ppm] Mult. δH [ppm] obs. signal pattern, J[Hz] 1 33.6 CH2 2.56/1.66 m 2 33.0 CH2 2.42/2.10 m/m 3 198.0 C - 4 123.6 CH 5.63 s 5 169.0 C - 6 31.4 CH2 2.43/2.26 m/d br (8.1) 7 31.8 CH2 1.90/1.22 m/m 8 35.6 CH 1.90 m 9 60.8 CH 2.12 d (11.5) 10 37.5 C - 11 210.4 C - 12 49.8 CH2 2.82/2.23 d (12.4) / d (12.4) 13 50.4 C - 14 48.9 CH 2.37 m 15 22.4 CH2 1.78/1,32 m /m 16 33.6 CH2 2,54/1.66 m /m 17 87.0 C - 18 14,9 CH3 0.50 s 19 16.4 CH3 1.31 s 20 207.0 C 21 84.1 CH 5.39 t br (7) 22 OH 5.66 s 23 OH 6.12* d br (7.8) 24 OH 6.36* d br (7.0)

Aldehyde form P Ia 20 200.5 C 21 195.2 CH 9.66 s, 2J(C,H) 27

* assignment may be interchanged

Table S3: 1H- and 13C-NMR data of P II in DMSO-d6

Pos. δC [ppm] Mult. δH [ppm] obs. signal pattern, J[Hz] 1 33.7 CH2 2.55/1.65 m/m 2 33.0 CH2 2.40/2.09 m/m 3 198.1 C - 4 123.3 CH 5.62 s 5 169.2 C - 6 31.5 CH2 2.42/2.25 m/d (7.9) 7 31.9 CH2 1.86/1.23 m/m 8 35.7 CH 1.86 m 9 60.7 CH 2.13 d (6.4) 10 37.6 C - 11 210.7 C - 12 50.1 CH2 2.76/2.14 d (12.4) / d (12.4) 13 51.0 C - 14 48.4 CH 2.25 m 15 22.8 CH2 1.73/1,22 m /m 16 32.0 CH2 2,55/1.47 m /m

17 87.0 C - 18 15.1 CH3 0.46 s 19 16.6 CH3 1.31 s 20 not obs. * C 21 not obs. * C - 22 OH not obs. 23 OH 7.01

* not observed due to limited amount of sample

Table S4: Metal content of homogeneous MroUPO

Metal Concentration Stoichiometry Method (µM) Metal/MroUPO* Mg 3.66 0.88 ICP-OES Mg 3.47 0.83 ICP-MS Fe 3.87 0.93 ICP-OES Mn 0.12 0.03 ICP-MS Cu 0,00 0.00 ICP-MS Zn 0.05 0.01 ICP-MS Mo 0.10 0.02 ICP-MS Pb 0.00 0.00 ICP-MS Ca 2.45 0.59 ICP-OES

* calculated for the monomeric MroUPO (32 kDa; 4.16 µM)

Figure S1: Time course of UPO production by Marasmius wettsteinii in a complex medium rich in C and N according to [2]; UPO activity (black circles), pH (dotted line). Culture flasks (500 mL) contained 200 mL medium and were shaken at 120 rpm. Data points represent mean values of three flasks with standard deviation.

Figure S2: HPLC-SEC analysis of different UPO preparations. A) MroUPO, B) MweUPO, C) AaeUPO and the respective UV-Vis spectra (insets). An Agilent 1200 HPLC system fitted with a Phenomenex Yarra 3µ SEC-2000 (300 x 7.8 mm) column was used; flow rate: 1 mL min-1, mobile phase: 100 mM Na acetate buffer + 100 mM NaCl (pH 6.9, T=25°C). Molecular weight standards for column calibration: gel filtration low molecular weight calibration kit (GE Healthcare). The insets show the characteristic UV- Vis absorption spectra of UPOs in their ground state with the Sorret band around 420 nm.

Figure S3: Denaturing (50 mM dithiothreitol) SDS PAGE (12 % NuPAGE Bis-Tris) of different fungal UPO preparations: 1) AaeUPO, 2) MweUPO, 3) MroUPO, 4) low molecular weight standard, Pierce™, Thermo Scientific, Darmstadt, Germany). The protein was visualized with a colloidal Blue staining Kit (Invitrogen, Carlsberg, USA).

Figure S4: HPLC-MS elution profiles of cortisone (S I) (0.5 mM) and its conversion products. Reaction -1 -1 solutions contained 1 U mL of MroUPO (I); H2O2 was supplied by syringe pump at 0.5 mM h . Insets show the respective MS spectra and structural formulae of substrate and products. P I – cortisone 21- gem-diol, P II – cortisone 21-oic acid, P III – adrenosterone.

Figure S5: HPLC-MS elution profiles on the basis of total ion counts (TIC; grey) of the conversion of cortisone by MweUPO. Samples were analyzed after 120 min (A) and 240 min (B). Extracted ion counts (EIC) for cortisone (S I, black) as well as of the products P I (blue), P II (red), P III (green), P IV (purpule) and P V (pink). The reaction solution contained 1 U mL-1 MweUPO and 0.5 mM cortisone (S I) in 10 mM

-1 potassium phosphate buffer (pH 5.5). H2O2 was supplied by syringe pump (0.5 mM h ). Note that the peak heights are relative and do not reflect real concentrations.

Figure S6: HPLC elution profiles of site chain cleavage products of Reichstein’s substance S (S II). I) Reaction solutions contained 1 U mL-1 MroUPO. II) carbonic acid, III) formic acid; separation was achieved on a Rezex™ ROA-Organic Acid H+ (8%) column (Phenomenex, Aschaffenburg, Germany).

whitish smear of preci- pitating BaCO3 MroUPO control

Figure S7: Reaction set-up of the BaCO3 precipitation experiment using Ba(OH)2 to trap CO2 formed during the reaction of MroUPO with cortisone.

Supplemental Information References

[1] V. Antonin, M.E. Noordeloos, A monograph of marasmioid and collybioid fungi in Europe, first ed., IHW-Verlag, Eching, 2010. [2] G. Gröbe, R. Ullrich, M.J. Pecyna, D. Kapturska, S. Friedrich, M. Hofrichter, K. Scheibner, High-yield production of aromatic peroxygenase by the fungus Marasmius rotula, AMB Express. 1 (2011) 31. [3] R. Ullrich, J. Nüske, K. Scheibner, J. Spantzel, M. Hofrichter, Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes, Appl. Environ. Microbiol. 70 (2004) 4575-4581. [4] M. Hofrichter, H. Kellner, M.J. Pecyna, R. Ullrich, Fungal unspecific peroxygenases: Heme-thiolate proteins that combine peroxidase and cytochrome P450 properties, in: E.G. Hrycay, S.M. Bandiera (Eds.), Monooxygenase, peroxidase and peroxygenase properties and mechanisms of cytochrome P450, Springer International Publishing, Cham, 2015, pp. 341-368.