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 48109. 1
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
Figure 3. MS and MS/MS spectra of mitomycin B (3). Figure 4. MS and MS/MS spectra of 7-demethylmitomycin A (4). 3
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. 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
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---------------------- 349 RdmB AGSAGLALASERTSGSTTLPFDFSILEFTAVSEEAAPAAQASEALPAQE 374 DnrK AASAGLVVEEVRQLPSPTIPYDLSLLVL-------APAATGA------- 360 CHOMT SKLSGFSKFQVACRAFNSLGVMEFYK----------------------- 372 COMT AKGAGFQGFKVHCNAFNTY-IMEFLKKV--------------------- 365 IOMT FIEAGFQHYKISP-LTGFLSLIEIYP----------------------- 352 :*: 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
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