Acyltransferase Domain Analysis using HMMER. Seoul National Univ. Hyo jin Kang
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1 Acyltransferase Domain Analysis using HMMER Seoul National Univ. Hyo jin Kang
2 Introduction What is an Acyltransferase? A group of enzymes catalysing the transfer of acyl groups Where can we find it? Capsaicinoid Biosynthetic Pathway in Pepper
3 Pungency of chili pepper Capsaicinoids major compounds of pungency placenta
4 Capsaicinoid Biosynthetic Pathway Phenylpropanoid Pathway Phenylalanine Cinnamic acid Coumaric acid Caffeic acid PAL Ca4H Ca3H COMT Branched-chain chain Fatty Acid Pathway Valine α-keto isovalerate Isobutyryl CoA Even Carbon Fatty acid L-leucine α-keto isocaporate isovaleryl CoA Odd Carbon Fatty acid L-isoleucine α-keto-β methylvalerate α-methyl-butyryl CoA Anti iso odd Carbon Fatty acid Ferulic acid Vanillin Vanillylamine pamt CS? Capsaicin Dihydrocapsaicin Nordihydrocapsaicin Homodihydrocapsaicin Homocapsaicin Homodihydrocapsaicin II Homocapsaicin II Nordihydrocapsaicin II
5 Finding cs gene Sequencing 80kb pepper Bac library Translating to amino acids with GENSCAN Contig13.fa
6 Acyltransferases domain search process 1. Query acetyltransferase in PROSITE 2. Select PDOC00402 Acyltransferases ChoActase / COT / CPT family signatures domain 3. ACYLTRANSF_C_1 Multiple-align 21 True positive hits 4. ACYLTRANSF_C_2 Multiple-align 25 True positive hits
7 ACYLTRANSF_C_1 CACP_COLLI (P52826), CACP_HUMAN (P43155), CACP_MOUSE (P47934), CACP_YEAST (P32796), CLAT_CAEEL (P32756), CLAT_DROME (P07668), CLAT_HUMAN (P28329), CLAT_MOUSE (Q03059), CLAT_PIG (P13222), CLAT_RAT (P32738), CPT1_HUMAN (P50416), CPT1_MOUSE (P97742), CPT1_RAT (P32198), CPT2_HUMAN (P23786), CPT2_MOUSE (P52825), CPT2_RAT (P18886), CPTM_HUMAN (Q92523), CPTM_RAT (Q63704), OCTC_BOVIN (O19094), OCTC_HUMAN (Q9UKG9), OCTC_RAT (P11466)
8 Multi-aligned sequences of ACYLTRANSF_C_1 OCTC_BOVIN20_35 OCTC_HUMAN20_35 OCTC_RAT20_35 CACP_YEAST74_89 CACP_HUMAN35_50 CACP_MOUSE35_50 CACP_COLLI36_51 CLAT_PIG23_38 CLAT_RAT22_37 CLAT_MOUSE23_38 CLAT_HUMAN131_146 CLAT_DROME88_103 CLAT_CAEEL20_35 CPT1_MOUSE162_177 CPT1_RAT171_186 CPT1_HUMAN171_186 CPT2_MOUSE49_64 CPT2_RAT49_64 CPT2_HUMAN49_64 CPTM_HUMAN173_188 CPTM_RAT173_188 LPSLPVPSLEESLKKY LPSLPVPSLEESLKKY LPPLPVPSLEESLKKY LPSLPVPELKSTLDKY LPRLPVPPLQQSLDHY LPRLPVPPLQQSLDYY LPHLPVPPLQQTLDRY LPKLPVPPLQQTLATY LPKLPVPPLQQTLATY LPKLPVPPLQQTLATY LPKLPVPPLQQTLATY LPKVPVPALDETMADY LPKPPVPSLEATLDRY LPRLPVPAVKDTVSRY LPRLPVPAVKDTVSRY LPRLPVPAVKDTVNRY LPRLPIPKLEDTMKRY LPRLPIPKLEDTMKRY LPRLPIPKLEDTIRRY LPKLPVPRVSATIQRY LPKLPVPSVPATIHRY
9 ACYLTRANSF_C_2 CACM_YEAST (P80235), CACP_CANTR (Q00614), CACP_COLLI (P52826), CACP_HUMAN (P43155), CACP_MOUSE (P47934), CACP_YEAST (P32796), CLAT_CAEEL (P32756), CLAT_DROME (P07668), CLAT_HUMAN (P28329), CLAT_MOUSE (Q03059), CLAT_PIG (P13222), CLAT_RAT (P32738), CPT1_HUMAN (P50416), CPT1_RAT (P32198), CPT2_HUMAN (P23786), CPT2_MOUSE (P52825), CPT2_RAT (P18886), CPTM_HUMAN (Q92523), CPTM_RAT (Q63704), OCTC_BOVIN (O19094), OCTC_HUMAN (Q9UKG9), OCTC_RAT (P11466), YB14_MYCPN (P75448), YEL4_YEAST (P40017), CPT1_MOUSE (P97742)
10 Multi-aligned sequences of ACYLTRANSF_C_2 CPT1_RAT451_478 CPT1_MOUSE442_469 CPT1_HUMAN451_478 CPTM_HUMAN451_478 CPTM_RAT451_478 CPT2_MOUSE350_377 CPT2_RAT350_377 CPT2_HUMAN350_377 OCTC_BOVIN305_332 OCTC_HUMAN305_332 OCTC_RAT305_332 CACP_CANTR314_341 CACP_YEAST356_383 CACP_HUMAN321_348 CACP_MOUSE322_349 CACP_COLLI322_349 CLAT_DROME397_424 CLAT_CAEEL319_346 CLAT_HUMAN420_447 CLAT_MOUSE312_339 CLAT_PIG312_339 CLAT_RAT311_338 CACM_YEAST325_351 YEL4_YEAST398_424 YB14_MYCPN301_328 RWFDKSITFV VFKNSKIGINA EHSWADA RWFDKSITFV VFKNSKIGINA EHSWADA RWFDKSFTFV VFKNGKMGLNAEHSWADA RWFDKSFTLI SFKNGQLGLNA EHAWADA RWFDKSFTLI SCKNGQLGLNT EHSWADA RWFDKSFNLI VAKDGTAAVHF EHAWGDG RWFDKSFNLI VAEDGTAAVHF EHSWGDG RWFDKSFNLI IAKDGSTAVHF EHSWGDG RWGDKSYNLIAFSNGVFGSNC DHAPFDA RWGDKSYNLISFSNGVFGCNC DHAPFDA RWGDKSYNLISFANGIFGCSC DHAPYDA RFFDKPLEFF VSANGNSGFLG EHSRMDA RFYDKSLQFL VTGNGSSGFLA EHSKMDG RWFDKTLQFI VAEDGSCGLVYE HAAAEG RWFDKTLQFI VAEDGSCGMVYEHAAAEG RWFDKTLQFI IAEDGSCGLVY EHAPAEG RWFDKTMQLIICTDGTWGLCYE HSCSEG RWYDATIQLVVSSSGVNGLCI E HSTAEG RWYDKSLQFVVGRDGTCGVVCEHSPFDG RWYDKSLQFVVGRDGTCGVVCEHSPFDG RWYDKSLQFVVGRDGTCGVVCEHSPFDG RWYDKSLQFVVGRDGTCGVVCEHSPFDG RWYDK-LQLIVTKNGKAGINFE HTGVDG RWYDK-LQLVVTADAKATVIWDSFTCDG RWHAKGIQLVITKNAQLVILAD HTSFDG
11 Making HMM model Acyltransf_C_1.hmm, Acyltransf_C_2.hmm hmmbuild Acyltransf_C_1.hmm Acyltransf_C_1.msf Calibration HMM model Hmmcalibrate Acyltransf_C_1.hmm
12 Further works Search SWISS-PROT database with HMM model Search contig13 sequence with HMM model Parsing the domain structure of a sequence with hmmpfam etc.
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