Supplementary Information. Autocatalytic backbone N-methylation in a family of ribosomal peptide natural products

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1 Supplementary Information Autocatalytic backbone -methylation in a family of ribosomal peptide natural products iels S. van der Velden 1, oemi Kälin 1, Maximilian J. Helf 1, Jörn Piel 1, Michael F. Freeman 2 * and Markus Künzler 1 * 1 Department of Biology, Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 893 Zurich, Switzerland 2 Department of Biochemistry, Molecular Biology, and Biophysics & BioTechnology Institute, University of Minnesota-Twin Cities, St. Paul, M 5518, USA *Co-corresponding authors *Correspondence: mkuenzle@ethz.ch (Markus Künzler) or mffreema@umn.edu (Michael F. Freeman).

2 Supplementary Results Supplementary Figure 1 SDS-PAGE page of purified proteins from E. coli and size exclusion chromatography of pha and pha-f. (a) SDS-PAGE of purified proteins from E. coli. 1 µg of each purified protein was run on a standard 12% (w/v) SDS-PAGE and stained with Coomassie brilliant blue. The name of the protein is followed by the duration of E. coli expression. Lane 1: pha, 24 hrs; Lane 2: pha, 5 days; Lane 3: DbphA, 24 hrs; Lane 4: DbphA, 5 days; Lane 5: pha-f, 5 days; Lane 6: pha-p, 5 days; Lane 7: pha-cyca, 5 days; Lane 8: pha-dica, 5 days; Lane 9: pha Y98A, 5 days; Lane 1: pha S129A (marked by ), 5 days. All proteins contain an -terminal histidine tag. Theoretical masses: pha, 46.9 kd; DbphA, 46.9 kd; pha-f, 46.3 kd; pha-p, 45.1 kd; pha-cyca, 45,7 kd; pha-dica, 46.8 kd. (b) Purified pha expressed for 24 hrs in E. coli BL21(DE3) eluted as high molecular mass aggregates (1st peak) and as a dimer (2nd peak) with an expected theoretical mass of 93.7 kd. (c) pha-f expressed for 24 hrs in E. coli BL21(DE3) mainly eluted as a dimer (2nd peak) with a theoretical mass of 92.5 kd. Insets: SDS-PAGE profile of the peak fractions. pha and pha-f are indicated with a. (d) The calibration curve used to estimate the observed molecular weight of pha and pha-f. The observed mass of the dimer of both pha and pha-f was 95.7 kd versus a theoretical mass of 93.7 kd for pha and 92.5 kd for pha-f. For calibration of the column the following size markers were used: carbonic anhydrase (29 kd), ovalbumin (44 kd), conalbumin (75 kd), ferritin (44 kd) and thyroglobulin (66 kd). The Y-axis represents the partition coefficient (Kav) and X-axis represents the log of the molecular weight (Mr). a kd 25 kd 13 kd 13 kd 1 kd 1 kd 7 kd 7 kd 55 kd 55 kd 2 kd 35 kd 35 kd 15 kd 25 kd 25 kd 15 kd 1 kd 75 kd 15 kd 1 kd 75 kd 5 kd 5 kd 37 kd 37 kd 25 kd 25 kd 2 kd 15 kd b 1 9 d c pha pha-f Carbonic Anhydrase 1 dimer 25kD 75kD aggregates 5kD 37kD 25kD Elution volume (ml) kD 75kD 5kD 37kD 1 25kD 5 valbumin dimer Kav 15 5kD 37kD UV absorption (mau) UV absorption (mau) 75kD 5kD 37kD aggregates.3 Conalbumin pha/pha-f.2 25kD.1 y= -.147ln(x) R2= Elution volume (ml) Mr Ferritin Thyroglobulin 1

3 Supplementary Figure 2 Protein sequence and structural alignments of the methyltransferase domain of pha. (a) Protein sequence alignment (ClustalW) along with secondary prediction (PSIPRED) of the methyltransferase domain of pha with homologous putative methyltransferases found in basidiomycota according to a BLAST search in the JGI fungal genome database ( Amino acids Y98 and S129 (highlighted in black) are fully conserved among all fungal homologues. The online software ESPript 3. was used to create the alignment figure. A red background denotes identical residues and red lettering denotes similar residues. The protein sequences corresponding to the respective protein ID numbers can be found on the JGI website ( programs/fungi/index.jsf). (b) The protein structural model of pha was generated using the online tool PHYRE2 28 and aligned with the bacterial homologues using PyML. Amino acid residues Y98 and S129 (black) of pha (red) are structurally conserved and lay in close proximity to the SAM binding pocket of the bacterial methyltransferases (magenta); cobalt-precorrin-4 (CobA) from Bacillus megaterium 29, dehydrogenase and ferrochelatase (CysG) from Salmonella enterica 3 and the SAM-dependent bismethyltransferase cytochrome cd1 nitrite reductase (ire) from Pseudomonas aeruginosa 31. Protein data bank files 1S4D (CobA), 1PJT (CysG) and 2YB (ire) were used for the generation of the structural alignments with pha.

4 Supplementary Figure 2 a Y98 b CobA S129A CysG ire S129 S129 S129 Y98 Y98 Y98

5 Supplementary Figure 3 Detected -methylations of pha-y98a and pha-s129a when expressed in E. coli alone or co-expressed with pha-v5t. Extracted ion chromatograms (EICs) of trypsin-treated, His-tagged (a) pha, (b) pha-y98a and (c) pha-s129a after five days of expression in E. coli compared to coexpression of pha-v5t with His-tagged (d) pha, (e) pha-y98a and (f) pha-s129a for 36 hrs. Methylation of pha-y98a and pha-s129a was only observed when coexpressed with pha-v5t, insinuating trans-methylation from pha-v5t. The catalytically active core mutant pha-v5t was used as a precaution for unambiguous detection of the pha-y98a and pha-s129a core peptides. Detected -methylated species are highlighted in green. Protein, time of expression in E. coli and HPLC retention time (RT) are listed in the upper right-hand corner of the spectrum. A mass window of.1 was used to create all EICs.

6 Supplementary Figure 3 a RT: min pha, 5 days [M+3H] 3+ ( ) [M+9Me+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) [M+11Me+3H] 3+ ( ) b RT: min pha-y98a, 5 days [M+3H] 3+ (126.58) 2 c [M+3H] 3+ (126.58) RT: min pha-s129a, 5 days

7 d RT: min pha, 36hrs [M+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) [M+2Me+3H] 3+ ( ) [M+3Me+3H] 3+ (14.512) [M+6Me+3H] 3+ ( ) [M+9Me+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) e RT: min pha-y98a, 36hrs [M+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) f RT: min pha-s129a, 36hrs [M+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) [M+1Me+3H] 3+ ( )

8 Supplementary Figure 4 Detected -methylations of pha after cell-free incubation. (a) EIC of purified pha after four hours of expression in E. coli. (b) EIC of purified pha (after four hours of expression in E. coli) incubated in an E. coli cell extract for three days at room temperature. Detected -methylated species are highlighted in green. Protein, time of expression in E. coli and HPLC retention time (RT) are listed in the upper right-hand corner of the spectrum. A mass window of.1 was used to create all EICs. More details concerning these data can be found in the methods. a RT: min pha, 4hr 1 8 [M+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) [M+2Me+3H] 3+ ( ) [M+3Me+3H] 3+ (14.58) b RT: min pha, 4hr, CF, 3 days [M+3H] 3+ ( ) [M+1Me+3H] 3+ ( ) [M+9Me+3H] 3+ ( ) [M+1Me+3H] 3+ ( )

9 Supplementary Figure 5 Structures of omphalotin B-I. Green depicts -methylations and blue denotes structural differences from omphalotin A. R 1 H R 2 H H R 3 H mphalotin B: R1 = H, R2 = mphalotin C: R1 = H, R2 = mphalotin D: R1 =, R2 = mphalotin E: R1 = H mphalotin F: R1 = H, R2 = H, R2 = H mphalotin G: R1 = H, R2 = H H, R3 = H, R3 = H, R3 = H, R3 = H, R3 = H, R3 = H H mphalotin H: R1 =, R2 = mphalotin I: H R1 =, R2 =, R3 =, R3 = H H omphalotins B-I

10 Supplementary Figure 6 pha is part of a gene cluster that is conserved in D. bispora. (a) The omphalotin gene cluster from. olearius and the homologous cluster found in D. bispora. Details regarding the individual genes in the clusters can be found in Supplementary Table 2. (b) Protein sequence alignment (ClustalW) along with secondary prediction (PSIPRED) of pha (JGI Prot. ID. 287) with DbphA (JGI Prot. ID ) using the online software ESPript 3.. A red background denotes identical residues and red lettering denotes similar residues. a. olearius omphalotin gene cluster ophb1 ophc opha ophd ophb2 ophp ophe dbophb1 dbophb2 dbophc dbophd1 dbophb3 dbopha dbophd2 dbophb4 dbophp dbophe 1 kb D. bispora putative borosin gene cluster Monooxygenase TF2-like Methyltransferase -acyltransferase Prolyloligopeptidase F-box/RI-like b pha pha DbphA pha pha DbphA pha pha DbphA pha pha DbphA pha pha DbphA core peptide

11 Supplementary Table 1 Primers used in this study

12 Supplementary Table 2 l Details of genes located in the. olearius and D. bispora borosin gene clusters *The protein sequences corresponding to the respective protein ID numbers can be found on the JGI website ( and /Denbi1/Denbi1.home.html). Supplementary References 28. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M.. & Sternberg, M. J. E. The Phyre2 webportal for protein modeling, prediction and analysis. at. Protoc. 1, (215). 29. Vévodová, J. et al. Structure/function studies on a S-adenosyl-L-methionine-dependent uroporphyrinogen III C methyltransferase (SUMT), a key regulatory enzyme of tetrapyrrole biosynthesis. J. Mol. Biol. 344, (24). 3. Stroupe, M. E., Leech, H. K., Daniels, D. S., Warren, M. J. & Getzoff, E. D. CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis. at. Struct. Biol. 1, (23). 31. Storbeck, S. et al. Crystal structure of the heme d1 biosynthesis enzyme ire in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-Lmethionine-dependent uroporphyrinogen III methyltransferases. J. Biol. Chem. 286, (211).

13 Supplementary ote: Mass spectra supporting the structural assignment of peptides described in this study.