Cytochrome P450 Monooxygenases

1
Cytochrome P450 Monooxygenases

• William Green
• Leland Taylor
• 3/29/11

2
Background

• P450s are a group of heme-containing enzymes that
  utilize NADPH and/or NADH to produce
  functionalized organic substrates
• Cytochrome P450-dependent monooxygenases mediate
  a wide range of oxidative reactions involved in
  the biosynthesis of plant secondary metabolites
  including phenylpropanoids and phytoalexins
  (Frank 1996).
• All P450s contain a highly conserved Amino Acid
  motif found near the C terminus F- -G-R-C-G
• Two main pathways of P450 monooxygenasese
• Biosynthetic Pathway
• Leads to syntheiss of UV protectants
  (flavonoids), anthocyanins, isoflavonoids/phytoale
  xins, fatty acids, hormones, signaling molecules,
  carotenoids, terpenes (defense compounds), and
  lignins (structural compounds)
• Detoxification Pathway
• Drugs, carcinogens, herbicides and insecticides
  are all detoxified by P450 controlled
  hydroxylations

3

• Goals of Study
• Identify Homologs of p450s in the Blueberry
  genome
• Create a network of pathways based on hits
• Find key p450s involved in secondary metabolites
• Ex. CYP73A5, CYP75A1, ect.
• Isolate exons introns
• Ideal World
• Annotate whole genes
• Link p450s with unknown functions to those with
  known functions
• Comparisons of dicot vs. monocot (Arabidopsis,
  rice, blueberry, etc.) in order to improve
  phylogenetic understanding

4
p450 hits against 454 database
Query Hit Subject Hit E Value Query Start Query End Subject Start Subject End
CYP89A7_NTC scaffold00007 7.00E-08 1303 1432 132874 132745
CYP89A7_NTC scaffold00007 3.00E-06 862 977 133386 133271
CYP89A7_NTC scaffold00007 4.00E-05 1207 1280 133008 132936
CYP89A9_NTC scaffold00007 3.00E-19 1300 1478 132877 132699
... ...
5
Isolate the sequence from scaffolds

• gtscaffold0225244524790CYP51G22e-40
• ACGTGGCCGGTCTTCGGCGGGTTAATCCGGTTCctAAAAGGaCCGGTTGT
  GATGCTAAGAGAAGAGTACCCGAAACTAGGGAGTGTGTTCACGTTGAACT
  TGTTCAACAAAAAGATCACTTTCTTTATCGGGCCTGAGGTTTCAGTCCAT
  TTCTTTAAAGCTTCTGAATCTAATCTGAGCCAACAAGAAGTGTACAAGTA
  CATTGTGCCTACCTTTGGGCCCGGAGTGATTTACGATGTTGATTATAGCG
  TACGGCAGGAGCAGTTTCGGTTCTTTACTGAGAGTCTAAGGGTTAACAAG
  TTGAAAGGTTATGTGGGTCAGATGCTTATTGAAGCTCA
• gtscaffold0225280638362CYP51G28e-35
• TCGCCTGCTTTCGCTAATCgTCTCCCTCATATATTCAAGGATCCGGACAC
  GTTTGACCCTGACAGATTTGCTCCCGGTAGGGAagAAGACAAAGTGTCTG
  GGGCgTTttCGTACATCTCTTTTGGCGGTGGCAGGCACGCGTGTCTAGGG
  GAGCCGTTCGCGTATCTGCAAATAAAATCgATTTGGAGCCATTTGCTGAG
  GAACTTTGAGCTGGAACTGGTTTCACCTTTTCCGGAGATTGACTGGAATG
  CCATGGTCGTGGGTGTGAAAGGAAaGgTTATGGTGCGGTACAAGCGCCG
• gtscaffold068661403014566CYP51G26e-16
• ACTGAATCTTTTTGTACTTCCAAATTACAGGACATGTTtGTGGGAGGAaC
  TGATACAACCTCAGCAACCATGGAGTGGGCAATGGCAGAGCTGATGAAAA
  aTCCAACAAaGATgAaGAAAGCTCAAAAaGAGGTGCGAAGAGTGGTTGGG
  AAAAAaTCCAAGAtcAGTCAAGATGATATTGCTCAAATGGAATACTTAAA
  aTGTGTTGTCAAAGAgACTCTTAGATTACATCCTCCAGCTCCTTTTTtGG
  TtCCTAGAGAATTGACTGAGAGTACTACATTGGAAGGCTATGATATTCCT
  CCAAAAaCAAGAATATTCATTAATGCATGGGCAATTCAAAGagATTCCAA
  ATACTGGGACCAGCCAGAAGAATTCcTTCCAGAGAGATTtGCTAACAACT
  TGGTTGATTTtAGAGGCCAAGAcTTCCAaTTCATTCCATTTGGAGCAGGG
  CgAAGGGGCTGTCCTGGCATTTCATTTGGTGTCATGGAAGTTGAACTTGT
  ACTTGCCAACCTCTTGTACTGGTTTGACTGGAAGTT
• gtscaffold0686692569750CYP51G26e-15
• GCAGGAACTGATACAACTTCAACAACCTtAGAGTGGGCAATGGCAGAGCT
  AATGAAAAGTCCAACTAGGATGAAGAAAaTtCAAGAaGAGGTACGAAGaG
  TGGTtGGgAAGAAaTCCAAGATCAATCAAgaTGATATtGATCAAATGgAC
  TACTTAAAATGTGTTGTCAAAGAGACTCTTAGATTACATCCTCCAGGTCC
  TTTTTtGGTtCCTAGAGAATCAAGTGAGAGTACTAAATTGGGAGGTTATG
  ATATTCCTCCAAAAACAAGAATTTTCATCAATGTTTGGGCAATTCAAAGA
  GATCCCAAATACTGGGATAGGCCCGAAGAATTTCTTCCAGAGAGATTTGC
  TAACAACCCGATTGATTTtAAAGGCAAAGACTTCGAATTCATCCCATTTG
  GTGCAGGGCGAAGGGGCTGTCCTGGAATTTCATTTGCAATCACTGAAACT
  GAACAAGTGCTTGCCAATCTCTTGTACTGGTTTGATTGGAAGTT

6
Align isolated sequences with EST

• gtscaffold0225244524790CYP51G22e-40
• ACGTGGCCGGTCTTCGGCGGGTTAATCCGGTTCctAAAAGGaCCGGTTGT
  GATGCTAAGAGAAGAGTACCCGAAACTAGGGAGTGTGTTCACGTTGAACT
  TGTTCAACAAAAAGATCACTTTCTTTATCGGGCCTGAGGTTTCAGTCCAT
  TTCTTTAAAGCTTCTGAATCTAATCTGAGCCAACAAGAAGTGTACAAGTA
  CATTGTGCCTACCTTTGGGCCCGGAGTGATTTACGATGTTGATTATAGCG
  TACGGCAGGAGCAGTTTCGGTTCTTTACTGAGAGTCTAAGGGTTAACAAG
  TTGAAAGGTTATGTGGGTCAGATGCTTATTGAAGCTCA
• Query scaffold0225280638362CYP51G28e-35
  gt Contig9662
• Query 4 CCTGCTTTCGCTAATCGTCTCCCTCATATATTCAAGGA
  TCCGGACACGTTTGACCCTGAC 63
• 
  
• Sbjct 582 CCTGCTTTCGCTAATCGTCTCCCTCATATTTTCAAGGA
  TCCGGACACGTTTGACCCTGAC 641
• Query 64 AGATTTGCTCCCGGTAGGGAAGAAGACAAAGT-GTCTG
  GGGCGTTTTCGTACATCTCTTT 122
• 
  
• Sbjct 642 AGATTTGCTCCCGGTAGAGAAGAAGACAAAGTAG-CTG
  GGGCATTTTCGTACATCTCTTT 700
• Query 123 TGGCGGTGGCAGGCACGCGTGTCTAGGGGAGCCGTTCG
  CGTATCTGCAAATAAAATCGAT 182
• 
  
• Sbjct 701 TGGTGGTGGCAGGCACGGGTGTTTAGGGGAGCCTTTTG
  CTTATCTGCAAATAAAAGCCAT 760
• Query 183 TTGGAGCCATTTGCTGAGGAACTTTGAGCTGGAACTGG
  TTTCACCTTTTCCGGAGATTGA 242
• 
  
• Sbjct 761 TTGGAGCCATTTGCTGAGGAATTTTGAGCTGGAACTGG
  TCTCACCTTTTCCGGAGATTGA 820

7
Issues/Frustrations

• gtscaffold0708653064950CYP51G26e-45
• Could not find good software
• JCVI, Washington University Genome Center
• Blast has tool to isolate poor documentation
• Artemis stinks