Cell Chemical Biology, Volume upplemental Information Investigation of Penicillin Binding Protein (PBP)-like Peptide Cyclase and ydrolase in urugamide on-ribosomal Peptide Biosynthesis Yongjun Zhou, Xiao Lin, Chunmin Xu, Yaoyao hen, hu-ping Wang, ongze Liao, Lei Li, ai Deng, and ou-wen Lin
upplementary Information Table of Contents Tables and Figures Table. Protein Analysis of sur BGCs Derived from LW0 and J0 Table. The ligonucleotide Primers Used in the tudy Table. The Plasmids Generated in the tudy Table. R-M Data of the Compounds in the tudy Figure. D-PAGE Gel of the Purified Proteins Figure. ure Active ite Prediction Figure. Protein equence Alignment of the ure omologues Data. QTF-M/M Analysis of the Compounds Generated in ure Assays Data. QTF-M/M Analysis of the ynthesized Peptidyl-AC ubstrates Data. and C MR pectra of the ynthesized Peptidyl-AC ubstrates
Table. Protein Analysis of sur BGCs Derived from LW0 and J0 (related to Figure ) Gene ize (a.a.) Predicted function Identity (%) Gene id in J0 urr GntR family DA-binding transcriptional regulator 00 XR orf Membrane protein 00 XR ure Beta-lactamase 00 XR0 ura RP (A-T, C-A-T-E, C-A-T-E, C-A-T) 99 XR9 urb RP (A-T, C-A-T-E, C-A-T, C-A-T) 00 XR urc 9 RP (C-A-T, C-A-T-E, C-A-T, C-A-T-E, C-A-T, C-A-T-E) 00 XR urd RP (C-A-T, C-A-T-E, C-A-T-E) 99 XR orf Drug resistance transporter 99 XR orf 0 TetR family transporter regulator 00 XR Table. The ligonucleotide Primers Used in the tudy (related to Figure and "ligonucleotides" in KEY REURCE TABLE) Primers equence Description In-frame deletion of sure gene lacd-l- GACTGATCAAGGCGAATACTTCAACTCCACGGTGCTCCTG 9 bp product, bp lacd-l-a lacd-r- lacd-r-a TAGTCGGAGTAGCTCTCGACGAACTGCATGACCAGTTC TGGTCATGCAGTTCGTCGAGAGCTACTCCGACTACCGCCTC TC AAAAGATCCGTCGACCTGCAGGCATGCAGCGTGTCGAGGG TGAC overlapping (L) with dei side of py bp product, bp L with indiii side of py, bp L with left fragment Identification of sure strain or complementation of sure strain with intact sure gene lac- ATATACATATGGGTGCCGAGGGGGCGGAAC bp for mutant, lac-a ATCGACCGCTCATCCTGGAACATCGTG 9 bp for wide type Complementation of sure strain with site-muated (A) sure gene The mutated gene was generated by way of overlapping PCR. MsurE- TTGTGGGCACAATCGTGCCGGTTG 0 bp MsurE-A TCACCGCGCCGAAGGGGAAACC MsurE- GTTTCCCCTTCGGCGCGGTGACCAAGTTCC bp MsurE-A TTCACACAGGAAACAGCTATGACATGATTACGAATTCGATATCTC Protein expression of ure with a -terminal is tag based on pet9a (dei, XhoI)
PsurE- PsurE-A ATATACATATGCATCACCACCATCATCATGGTGCCGAGGGG GCGGAAC AATATCTCGAGTCAGAGCCGGTGCATGGCCCGGCCGCCGT Protein expression of ure with A site mutation MurE- A-A A- MurE-A AATTTTGTTTAACTTTAAGAAGGAGATATACATATGCATCACCACCATC AGGAACTTGGTCACCGCGCCGAAGGGGAAACCCGTC TTCCCCTTCGGCGCGGTGACCAAGTTCCTCACCGCCGAAC TTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTC Protein expression of ure with KA site mutation MurE- KA-A KA- MurE-A see above TGAGGAACGCGGTCACCGAGCCGAAGG CTTCGGCTCGGTGACCGCGTTCCTCACCGCCGAACTGGTC see above Protein expression of ure with YA site mutation MurE- YA-A YA- MurE-A see above TGTTGGACGCGGAGAAGGCGAGGCCCGGCGGGA CTCGCCTTCTCCGCGTCCAACACCGGGTACTGCCTG see above Protein expression of ure with A site mutation MurE- A-A A- MurE-A see above AGGCAGTACCCGGTCGCGGAGTAGGAGAAGGCGAGG CTACTCCGCGACCGGGTACTGCCTGCTGGGCGCGGT see above 9 bp product with dei and XhoI sites Table. The Plasmids Generated in the tudy (related to Figure and "Plasmid Constructions" in the section of TAR METD) Plasmids Descriptions In-frame deletion of sure gene in sur BGC prj Two PCR products were amplified from the genomic DA of LW0 with the primers lacd-l- and -A for the left fragment, and lacd-r- and -A for the right fragment, respectively (Table ). The two PCR products were assembled with the vector fragment of py (dei, indiii) by the way of Gibson assembly. Complementation of sure strain prj9 prj A PCR fragment of sure amplified from the genomic DA of LW0 with the primers, lac- and-a (Table ), was digested with dei and EcoRV and then ligated into pib9 (dei, EcoRV) by T ligase. Two PCR products amplified from prj9, respectively with the pairs of primers MsurE- and MsurE-A, and MsurE- and MsurE-A (Table ), were assembled into one fragment. The fragment was then introduced into pib9 (dei, EcoRV) by the way of Gibson assembly.
Protein expression of ure with -terminal is tag in E. coli BL(DE) plys. prj Wide type ure The PCR product of sure amplified from prj9 with the primers, PsurE- and -A (Table ), was digested with dei and XhoI, followed by ligation into the corresponding site of pet9a prj0 ite mutation (KA) The two PCR products (see primers in Table ) of prj ite mutation (A) sure containing the target mutation site at overlapping region were introduced into pet9a prj ite mutation (YA) (dei, XhoI) by using Gibson assembly. prj ite mutation (A) prj9 ite mutation (A) The synthesized DA fragment of sure with target prj ite mutation (DA) mutation was introduced into pet9a (dei, XhoI). prj ite mutation (D) Table. R-M Data of the Compounds in the tudy (related to Figure and Table ) Products Formula calc. m/z det. m/z Error (ppm) The compounds produced in enzymatic assays or by strain LW0 C 9 9. 9.. ~ C 9 9 9.0 9.0 -. C 0. 0.9 -. C 9 9 90. 90.9 0. 9 C 0 9 9. 9.. 0 C 9 9 9.0 9.. C 9... C 0. 0.9 0. The substrates used in enzymatic assay 0 C 90 0 9 0. 0. 0. C 90 0 9 0. 0.. C 0 9 0.9 0.9.9 C 9 0 9 99. 99. 0.9 C 0 9 9.00 9.09 0. C 9..0.
D-PAGE gel of ure protein purification The concentration of imidazole in elution (mm) 0 0 0 0 0 00 0 00 FT pellet. kd kd kd kd kd Figure. D-PAGE Gel of the Purified Proteins (related to Figure ) Putative key residues in the active site key residues in the active site key residues in the active site Tyr is Asp Lys er Asn Predicted ure overall structure ADP overall structure DAP overall structure Figure. ure Active ite Prediction (related to Figure and Figure ) tructural and Active ite Comparison among ure, ADP (PDB id YP) and DAP (PDB id EI_A). The proposed active sites and structure of ure is predicted based on the site-mutation and the comparative analysis.
() 0 0 0 0 0 0 0 0 90 00 0 0 cmcpbp () --------------------------------------------MPFDAWQERFDALRTEVPGAALAVFVDG-DLELAGVLRGTGVAVTTDVFQGVAKVYTATLVMQLVDAGELRLDTRVADVL Pab () --------------------------------------EQUECEMDVGKLEFIVEKMAERKVPGIIIIKDGDVVYAKGFGYRVEARLPTPETIYGIGITKFTALAIMKLVEEGGLLDDPVEKFV ADP () MKTRQITCALALLIAGLLYTTQTLIVKAEPTQVVQTTQRDRVKQAVRDTLQLGFPGILAKTEGG-KTWYAAGVALKKPMKTDFRFRIGVTKTFTATVVLQLAEERLLDDIEKWL DAP () -------------------------------------------MKFDTALEAFVRIPQYKGPGGVVAVVKDGEVVLQAWGFADLRTRTPMTLDTRMPICVKQFTCAVLLDAVGE-PELLDDALEAYL MppK () ----------------------------------------------MDELTLTELAARRVPGAQLAVIEG-ERFLVTGVCDTAGAPVERTAFPVGLTKPFTAALAMILVADGDVDLDEPLRGQL DsaJ () -------------------------------MLGIIAEEKVGAPFLPLDRLGCLGLGKEGVVGAQLAVYRG-TLRTWEFGEEEYGGGRPVRRDAFPYGVTKALTAAVLQLVDGDLDLRPVRTWL Ulm () -------------------------------MGDYADPADCGAGDGAPVG--LDLDRLARDCDVVGGQLALQG-TLTTWEFGTEEAGGRPVVGAFPYGVTKAFTATAVLQLAGDGDLDLDRPVRELL ure () -----------------------------------------VGAEGAERDAVGALFEELVRERVTGAQLAVYRDG-ALEYATGLAVRTGEPVTPRTGFPFGVTKFLTAELVMQFVCDGDLDLDDPLAGLL Consensus () D L A L L EKVPGAQLAV DG L WAFGV DLATG PVT DTAFPIGVTK FTA LVLQLV DGDLDLDDPV WL xxk K () 0 0 0 0 0 90 00 0 0 0 0 0 cmcpbp (90) PGFA----------------VADAEVARTVTIGRLLTGIAGDFTLDTGRG-DDCLARFVDACADVG-----------QDCPPDTVIYCTGYAILGRIVEVLTGQWDDALRDRLFTPLGLQMTLP-- Pab (9) IKLR-------------------PFGEPVTVLLTGIPLGYAEAFIDGMVGGDWLPVTPEETIAFARDMEKWAVAKPGERFFYLTGYVLLGKIIEKVGVYEEYIKKKILEPLGMRYFFK-- ADP() PGVIQ----------------GGYDDKQITIRQLLTGIAEYTRKFDLMDTKKYRAEELVKMG-IMP------PDFAPGKWYTGYVLLGILIETVTGYAEEIERIIEPLELTFLPG- DAP (9) DKFED---------------------ER-PAVRDLCQGLRDYWALVLCGADPEGVFLPAQAQLLRRLKT------TFEPGYYCGFRILADLIEATGRTLVDILERIFAPAGMKRAELI-- MppK () PEFG------------------AGELVT---LRQLLTGLPDVPEGDEAGGGDRARWVARYCRTAD----------LTAPGTVFYIGYVVVGRLIEAVTGMWQEAIAILLEPLGTRPAFVVG-- DsaJ(0) DDRG----------------AAGPGLGATLRQLLTAGLP----DDDGDATLRRWLTGFLATGPADAP------ALWPAPGFYVGYGIAGRVVEAATGLTWWQAVRDYLLAPLGTGIGLLPGAP Ulm(0) PEAEAEAEIEVEAGGTGARADGGPALAATLRQLLTAGLP----DDDERAPLRRWLTGFLALP----V------GPWPAPGFYVGYGIAGRVVEAVTGLTWEAVRDFLLPLGTAITVLP--- ure (9) PDLG----------------RAAGPALGTATVRQLLTAGVVDI--EYDEMRGPYRRFAAACARQP-----------ALFPPGLAFYTGYCLLGAVIEAAGMDWWTAMDCLLRPLGIEPAFLDP- Consensus() PD G AAG A TLRQLLTGLP D DDG A RWL A LG A FPPG FYTGY ILGRLIEAVTGLW EAIRDRLL PLGL AFL Yx Y () 0 90 00 0 0 0 0 0 0 0 0 90 0 cmcpbp(9) EEALRFRVAMLGELGTDPEPAPVWDMLPRAGPYGRVLITAADVVRFARMLDDGVAPDGTRVLAAAALMRQQVAGCLDTWFMATGWGGWALYDWD-GVPGYGDGAGGQFYLRVVPGGVVAALL Pab(0) --EEVEKDKDVAMGYILDK-EGRLVPQPFPYGITADGGLLVLDLAKYLKMYIERDEIV------KEYIEKMETYIKVPWEIFGGEGYGYGLIIYPFLGEKLVGGVGMYTGYIGYIPEKKIGVAVL ADP() VIPGTKARGYIQLDGAEPKDVTYYPMGAGDMITADDLKFFYLLGGKLLKE-----------QQLKQMLTTVPTGEAALGRYGLGIYETKLPGVIWGGGIPGFVTFAGGTLGGKTLAV DAP(9) DTALFDECTG-----YEGDTVRGFLPATRIQWMGDAGICALDMIAWEQFIDATRDDEG-----------LYRRLGPQTFKDGVAAPYGFGLLETG-GKRLTGGGALRGWRCQRWCADERLTIAM MppK(9) -APATRPVATGAVQAVRDRVVPIPDQDLPEVEMPGALALAEDLVGFARLYFAGCPDPQPLDRATADDMCFDQLAIAIGPYG--MADGWGLGWARFDDG-AADVYGGTGDGTCLRFDPAGAVALT DsaJ() EPVRAVAGTTTTPGG-DRVVVRCADAGFTPAGGLAGAADLVRFAQLLDDPGDLDRDAVADETLREMARPVAGAEPFG--LADGWGLGLGFGPA-EDRWLGDGTLDGTTCLRIPKQGTVVALT Ulm() ----------------------------TDPGLPAGGLAGAADLVRLGRLLDEPGDPDLARLADPDALREMARPTAGADPFG--LADGWGPGLGRFGPA-GRWLGDGTLDGATCLRIPGRGTVVALT ure(00) QGGAARPVAEGALRAG-GERAERVDMALLAAAGGLVGATDLVTAARPLADRKTFAQDLLPEDAVLAMRTCVPDAEPFG--LADGWGLGLMRGTG-DGAWYGDGAVGGACLRIPDRLALALT Consensus() D V P PAGGLLGA DLVRFARLLDD D D L D L M R VAG PFG LADGWGLGLA F G G RLYGDGTL G TCLRI PG GL VALT (0) 0 0 0 0 0 0 0 0 90 00 0 0 9 cmcpbp() TGGVATFFTDLFRELLGELAGVRMPEVFAPPAEPRPIDVAPLAGTYE--REGGAP----------------------------------------------------------------------------- Pab() EG----YPPYIAMYALALLLGKPEKELPFIYRERILKKVEGRYMGYKGTIKFEVKVDGDVVYLRALGRAFTYTIPLFPEVLEEDFIKCYTLGRKMYAEFYIKDKVDLIFERYRLIK--------- ADP() LLAEPDPFKILLAEFK---------------------------------------------------------------------------------------------------------------- DAP() FFEGGAEVAFKLMIALGV-EVRVEADAWFGWLDDETGLVLLEDAGGRMKARFGTPEMMDVVAEARAVTTIRRDGETIELVRAELRLMKRVKGEAKDIIGRYDELDADLLLVE MppK(9) AATGAQLWDALVPRLRAMGLAVGDRPAPEPPTTP-PPVPDDCPGRYT--GDTEFVVQPGADGGLLLFGGAPELLCPDLRFTMRELG-GARPGRFVTDPATGRIGYLQITGRLAPRR--------- DsaJ() TAGQGLWEAVLRELRAGLPVGVYVVPPPG-TAGFADCAGTYR--GDLAVTVLDDR-ILLELPGARDAADLVFRPGD--AGVFLGRFLKDATTGDVRVLQYGRTLLRQP---VAAQP Ulm() TPTGQALWDAVVDALRDADIDVGVRPAPPPAIA-AAAFADCTGTYR--GDLAVTVGIDGP-YLVLELPGGARELAQPLARTFR-----GAGFLGRFVTDARDAVALQYGRTLLREAGERRAR ure(0) ATAGPKLWEALVARLPEAGLDVGYALPVPDAP-LAPDAGLGTYA--GDLELMVTDAAGDLFLTREYDYRLLEDDLFVARGEPGALPITGRFVREPAGPVALLQYGGRAMRL--------- Consensus(0) G LWDALV L AGL VG PPA A AD GTY GDL V DA LLL AA DL F R AA GRFV D G V LQY GR L R DG D Figure. Protein equence Alignment of the ure omologues (related to Figure and Figure ) The putative activities are labeled. The site mutations generated in ure are displayed with the AA sequence number. Data. QTF-M/M Analysis of the Compounds Generated in ure Assays (related
to Figure ).. The M fracture spectrum of the compound generated by ure with compound 0 as substrate (a) and the one isolated from the fermentation extract of LW0 (b). The above scheme is the predicted fragments of the M/M analysis. Chemical Formula: C 9 Exact Mass: 9. Chemical Formula: C 9 Exact Mass:. compound 9 Chemical Formula: C0 Exact Mass:.0 Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C Exact Mass: 99.0 Chemical Formula: C Exact Mass:.09 0 Chemical Formula: C9 Exact Mass:.09 Chemical Formula: C9 Exact Mass:.9 Chemical Formula: C Exact Mass:. Chemical Formula: C0 Exact Mass:.00 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass:.9 a) 0 9 b).. The M fracture spectrum of compound produced by J0 (a) or the one generated in ure assay with compound as substrate (b). The above scheme is the predicted fragments of the M/M analysis.
Chemical Formula: C Exact Mass: 0. Chemical Formula: C 9 9 0 0 Exact Mass: 9.9 Chemical Formula: C 0 9 9 Exact Mass:.9 compound Chemical Formula: C Exact Mass: 9.90 Chemical Formula: C 9 Exact Mass:. Chemical Formula: C Exact Mass: 99.9 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass: 00.0 a) b).. The M fracture spectrum of compound generated in ure assay with compound 0 as substrate. The above scheme is the predicted fragments of the M/M analysis. Exact Mass: 90. compound Chemical Formula: C99 Chemical Formula: C Exact Mass: 99.0 Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C9 Exact Mass:.9 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:... The M fracture spectrum of compound 9 produced in ure assay with compound as substrate. The above scheme is the predicted fragments of the M/M analysis.
Chemical Formula: C09 Exact Mass: 9. compound 9 Chemical Formula: C9 Exact Mass:. Chemical Formula: C Exact Mass:.9 Chemical Formula: C9 Exact Mass: 0. Chemical Formula: C9 Exact Mass: 0. Chemical Formula: C0 Exact Mass:.00 9 0 Chemical Formula: C Exact Mass: 9.0 Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C0 Exact Mass:. Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:.09 Chemical Formula: C0 Exact Mass: 0. 0 9.. The M fracture spectrum of compound 0 produced in ure assay with compound as substrate. The above scheme is the predicted fragments of the M/M analysis. Chemical Formula: C99 Exact Mass: 9.0 compound 0 Chemical Formula: C Exact Mass: 99.0 Chemical Formula: C Exact Mass: 0.0 Chemical Formula: C Exact Mass:. Chemical Formula: C0 Exact Mass:. Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C9 Exact Mass:.9 9 Chemical Formula: C09 Exact Mass:.0 Chemical Formula: C Exact Mass: 9. 9.. The M fracture spectrum of compound produced in ure assay with compound as substrate. The above scheme is the predicted fragments of the M/M analysis.
Chemical Formula: C9 Exact Mass:. compound Chemical Formula: C9 Exact Mass:.9 Chemical Formula: C0 Exact Mass:.00 Chemical Formula: C Exact Mass:.9 9 Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 0. Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 0. 9.. The M fracture spectrum of compound produced in ure assay with compound as substrate. The above scheme is the predicted fragments of the M/M analysis. Chemical Formula: C Exact Mass: 0. compound Chemical Formula: C 9 0 9 Exact Mass: 99.0 Chemical Formula: C099 Exact Mass:.9 Chemical Formula: C0 Exact Mass: 9.9 9 Chemical Formula: C9 Exact Mass: 9.0 Chemical Formula: C0 Exact Mass: 0. Chemical Formula: C0 Exact Mass: 9. Chemical Formula: C Exact Mass: 0. Chemical Formula: C Exact Mass:. 9
Data. QTF-M/M Analysis of the ynthesized Peptidyl-AC ubstrates (related to Figure ).. The M fracture spectrum of compound compounds 0 (a) and (b). The above scheme is the predicted fragments of the M/M analysis. Chemical Formula: C909 Exact Mass: 0. Chemical Formula: C9 Exact Mass: 9. compound 0 or Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 99.0 Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass:.9 Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C9 Exact Mass:.9 9 0 Chemical Formula: C09 Exact Mass:.0 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:. Chemical Formula: C9 Exact Mass:. a) 0 9 b).. The M fracture spectrum of compound compounds. The above scheme is the predicted fragments of the M/M analysis.
Chemical Formula: C90 9 Exact Mass: 0.9 Chemical Formula: C09 Exact Mass: 9. compound Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 99.0 Chemical Formula: C0 Exact Mass:. Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 9.9 9 0 Chemical Formula: C Exact Mass:. Chemical Formula: C9 Exact Mass:.9 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:. 0 9.. The M fracture spectrum of compound compounds. The above scheme is the predicted fragments of the M/M analysis. Chemical Formula: C90 9 Exact Mass: 99. Chemical Formula: C9 Exact Mass: 0. compound Chemical Formula: C Exact Mass: 99.0 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:. Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass: 9.9 Chemical Formula: C9 Exact Mass:.9 9 0 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass:. Chemical Formula: C9 Exact Mass:. 9 0.. The M fracture spectrum of compound compounds. The above scheme is the
predicted fragments of the M/M analysis. Chemical Formula: C09 Chemical Formula: C9 Chemical Formula: C9 Chemical Formula: C09 Exact Mass: 9.00 Exact Mass:. Exact Mass:. Exact Mass: 0.00 compound Chemical Formula: C Exact Mass:. 9 0 Chemical Formula: C0 Chemical Formula: C9 Chemical Formula: C Chemical Formula: C Exact Mass: 0. Exact Mass: 9.0 Exact Mass: 9. Exact Mass: 9.9 Chemical Formula: C0 Exact Mass: 0.0 Chemical Formula: C Exact Mass:. 0 9.. The M fracture spectrum of compound compounds. The above scheme is the predicted fragments of the M/M analysis. Chemical Formula: C9 Exact Mass:. compound Chemical Formula: C099 Exact Mass:.9 Chemical Formula: C Exact Mass: 9. Chemical Formula: C Exact Mass: 0. Chemical Formula: C Exact Mass: 9.90 Chemical Formula: C0 Exact Mass:.9 Chemical Formula: C9900 Exact Mass: 9.9 Chemical Formula: C 9 Exact Mass: 9. Chemical Formula: C0 Exact Mass: 0. 9 0 Chemical Formula: C Exact Mass: 99.9 9 0
Data. and C MR pectra of the ynthesized Peptidyl-AC ubstrates (related to Figure ).. - (a) and C- (b) MR spectrum of 0 a)
b).. - (a) and C- (b) MR spectrum of a)
b).. - (a) and C- (b) MR spectrum of a) b)
.. - (a) and C- (b) MR spectrum of a) b)
.. - (a) and C- (b) MR spectrum of a) b)
.. - (a) and C- (b) MR spectrum of a) b)