Hydroxyquinone O-Methylation in Mitomycin. Biosynthesis

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Maria Danielsson
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1 Supporting Information Hydroxyquinone O-Methylation in Mitomycin Biosynthesis Sabine Grüschow, Leng-Chee Chang, Yingqing Mao, and David H. Sherman Life Sciences Institute, Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI

2 Mass Spectrometrical Data for 1-6 Figure 1. MS and MS/MS spectra of mitomycin C (1). Figure 2. MS and MS/MS spectra of mitomycin A (2). 2

3 Figure 3. MS and MS/MS spectra of mitomycin B (3). Figure 4. MS and MS/MS spectra of 7-demethylmitomycin A (4). 3

4 Figure 5. MS and MS/MS spectra of 7-demethylmitomycin B (5). Figure 6. MS and MS/MS spectra of albomitomycin A (6). 4

Southern Blot Hybridization 5.8 kb 3.3 kb 1.3 kb Figure 7. Southern blot hybridization. A) DHS9505: mmcr deletion. B) mmcr::aphii. C) S. lavendulae wild-type. M) Marker.

5 Southern Blot Hybridization 5.8 kb 3.3 kb 1.3 kb Figure 7. Southern blot hybridization. A) DHS9505: mmcr deletion. B) mmcr::aphii. C) S. lavendulae wild-type. M) Marker. The expected hybridization bands were 3.1 kb (A), 4.9 kb (B), 3.5 kb (C) for SacI digestion; 3.3 kb (A), 5.8 kb (B), 4.4 kb (C) for BamHI digestion; and 3.8 kb (A), 1.3 & 4.9 kb (B), 1.3 & 3.5 kb (C) for EcoRI digestion. MmcR Sequence Analysis Figure 8. Phylogenetic tree (unrooted) of MmcR homologues. The tree was constructed with Phylip 3.6 using the maximum likelihood method. 1 Accession numbers for the individual proteins are: IOMT: O24529, COMT: P28002, CHOMT: P93324, DnrK: Q06528, RdmB: Q54527, MmcR: Q9X5T7, PurMT: Q090M0, SfcG: Q5JCL4, LipMT: Q30CS5, GilMT: Q7X2G8, PrdMT: O32456, FrdMT: Q2MGC2, OxyF: Q3S8Q9, CalO1: Q8KNE5, Asm7: Q8KUF3, KanP: Q65CE6, CalO6: Q8KND2, OxyT: Q3S8P6, MtmMI: Q194P7. The sequence identities with respect to MmcR are indicated for each protein. 5

6 MmcR ----MTVEQTPENPGTAARAAAEETVN--DILQGAWKARAIHVAVELGVPELLQEGPRTATALAEA TGAHEQTLRRLLRLLATVGVFD------DLGHDDLFAQNALSA 97 RdmB -----MSSSSPGEP-LEPTDQDLDVL--LKNLGNLVTPMALRVAATLRLVDHLLAGADTLAGLADR TDTHPQALSRLVRHLTVVGVLE------GGEKQGRPLRPTRLG 95 DnrK -----GSPNSTAEPTVAARPQQIDALRTLIRLGSLHTPMVVRTAATLRLVDHILAGARTVKALAAR TDTRPEALLRLIRHLVAIGLLE------EDAPG--EFVPTEVG 96 CHOMT MGNSYITKEDNQISATSEQTEDSACLSAMVLTTNLVYPAVLNAAIDLNLFEIIAKATPPGAFMSPSEIASKLPASTQHSDLPNRLDRMLRLLASYSVLTSTTRTIEDGGAERVYGLSMVG 120 COMT MG----STGETQITPTHISDEE-ANLFAMQLASASVLPMILKSALELDLLEIIAKAG-PGAQISPIEIASQLP--TTNPDAPVMLDRMLRLLACYIILTCSVRTQQDGKVQRLYGLATVA 112 IOMT MASSINGRKPSEIFKAQALLYKHIYAFIDSMSLKWAVEMNIPNIIQNHGKPISLSNLVS------ILQVPSSKIGNVRRLMRYLAHNGFFE------IITKEEESYALTVAS 100. :. * : : : :. : *::* *..: :. MmcR VLLPD--PASPVATDARFQAAPWHWRAWEQLTHSVRTGEAS-FDVANGTSFWQLT--HEDPKARELFNRAMGSVSLTEAGQVAAAYD-FSGAATAVDIGGGRGSLMAAVLDAFPGLRGTL 211 RdmB MLLADGHPAQQRAWLDLNGAVSHADLAFTGLLDVVRTGRPA-YAGRYGRPFWEDLS--ADVALADSFDALMSCDEDLAYEAPADAYD-WSAVRHVLDVGGGNGGMLAAIALRAPHLRGTL 211 DnrK ELLADDHPAAQRAWHDLTQAVARADISFTRLPDAIRTGRPT-YESIYGKPFYEDLA--GRPDLRASFDSLLACDQDVAFDAPAAAYD-WTNVRHVLDVGGGKGGFAAAIARRAPHVSATV 212 CHOMT KYLVPDESRGYLASFTTFLCYPALLQVWMNFKEAVVDEDIDLFKNVHGVTKYEFMG--KDKKMNQIFNKSMVDVCATEMKRMLEIYTGFEGISTLVDVGGGSGRNLELIISKYPLIKGIN 238 COMT KYLVKNEDGVSISALNLMNQDKVLMESWYHLKDAVLDGGIP-FNKAYGMTAFEYHG--TDPRFNKVFNKGMSDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINTIVSKYPTIKGIN 229 IOMT ELLVRG-SDLCLAPMVECVLDPTLSGSYHELKKWIYEEDLTLFGVTLGSGFWDFLD--KNPEYNTSFNDAMASDSKLINLALRDCDFVFDGLESIVDVGGGTGTTAKIICETFPKLKCIV 217 * : : :. : :. *. *. : : : : :*:*** * : * : MmcR LERPPVAEEARELLTGRGLADRCEILPGDFFETIPDGADVYLIKHVLHDWDDDDVVRILRRIATAMKPDSR---LLVIDNLIDERPAASTLF-----VDLLLLVLV-GGAERSESEFAAL 322 RdmB VELAGPAERARRRFADAGLADRVTVAEGDFFKPLPVTADVVLLSFVLLNWSDEDALTILRGCVRALEPGGR---LLVLDRADVEGDGADRFF--STLLDLRMLTFM-GGRVRTRDEVVDL 325 DnrK LEMAGTVDTARSYLKDEGLSDRVDVVEGDFFEPLPRKADAIILSFVLLNWPDHDAVRILTRCAEALEPGGR---ILIHERDDLHENSFNEQF---TELDLRMLVFL-GGALRTREKWDGL 325 CHOMT FDLPQVIENAPPLSG IEHVGGDMFASVPQ-GDAMILKAVCHNWSDEKCIEFLSNCHKALSPNGK---VIIVEFILPEEPNTSEESKLVSTLDNLMFITV-GGRERTEKQYEKL 346 COMT FDLPHVIEDAPSYPG VEHVGGDMFVSIPK-ADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGK---VIVAECILPVAPDSSLATKGVVHIDVIMLAHNPGGKERTQKEFEDL 338 IOMT FDRPQVVENLSGSNN LTYVGGDMFTSIPN-ADAVLLKYILHNWTDKDCLRILKKCKEAVTNDGKRGKVTIIDMVIDKKKDENQVTQIKLLMDVNMACLN--GKERNEEEWKKL 327.:. : : **:*.:*.*. ::. : :* *.. : :* *:..: : : :. : * *...: * MmcR LEKSGLRVERSLPCGAGPVRIVEIRRA RdmB AGSAGLALASERTSGSTTLPFDFSILEFTAVSEEAAPAAQASEALPAQE 374 DnrK AASAGLVVEEVRQLPSPTIPYDLSLLVL APAATGA CHOMT SKLSGFSKFQVACRAFNSLGVMEFYK COMT AKGAGFQGFKVHCNAFNTY-IMEFLKKV IOMT FIEAGFQHYKISP-LTGFLSLIEIYP :*: Figure 9. Sequence alignment of MmcR to AdoMet-dependent enzymes with known structure. RdmM: aclacinomycin 4- hydroxylase from Streptomyces purpurascens, PDB 1R00; 2 DnrK: carminomycin 4-O-methyltransferase from Streptomyces peuceticus, PDB 1TW3; 3 CHOMT: isoliquiritinenin 2 -O-methyltransferase from Medicago sativa, PDB 1FP1; 4 COMT: caffeic acid 3-O-methyltransferase from M. sativa, PDB 1KYZ; 5 IOMT: isoflavone 7-O-methyltransferase 8 from M. sativa, PDB 1FP2. 4 Catalytic residues (yellow) and amino acids involved in AdoMet (orange) or substrate (green) binding are highlighted. The alignment was generated using ClustalW. 6 6

7 References 1. Felsenstein, J. Cladistics 1989, 5, Jansson, A.; Koskiniemi, H.; Erola, A.; Wang, J.; Mantsala, P.; Schneider, G.; Niemi, J. J. Biol. Chem. 2005, 280, Jansson, A.; Koskiniemi, H.; Mantsala, P.; Niemi, J.; Schneider, G. J. Biol. Chem. 2004, 279, Zubieta, C.; He, X. Z.; Dixon, R. A.; Noel, J. P. Nat. Struct. Biol. 2001, 8, Zubieta, C.; Kota, P.; Ferrer, J. L.; Dixon, R. A.; Noel, J. P. Plant Cell 2002, 14, Thompson, J. D.; Higgins, D. G.; Gibson, T. J. Nucleic Acids Res. 1994, 22,

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SUPPLEMENTARY FIGURE LEGENDS

SUPPLEMENTARY FIGURE LEGENDS SUPPLEMETARY FIGURE LEGEDS Supplementary Fig. 1. Flow cytometric analysis of wildtype, mutant and chimeric protein surface expression. Cells transduced with the individual constructs indicated were stained