Phylogenetic analysis of Cytochrome P450 Phylogenetic analysis of Cytochrome P450 Structures Structures Gowri Shankar, University of Sydney, Australia. Gowri Shankar, University of Sydney, Australia.
Overview Overview Introduction Introduction Cytochrome P450 -- Structures. Cytochrome P450 -- Structures. Results. Results. Conclusion. Conclusion. Future Work. Future Work. Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Aims Aims How the structure of the cytochrome P450 changes How the structure of the cytochrome P450 changes – Time Time – – Across species. Across species. – Estimate the ages and functional Changes with structural changes. Estimate the ages and functional Changes with structural changes. How the residues changes with time and its impact on structure and How the residues changes with time and its impact on structure and function of CYPs. function of CYPs. Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Introduction Introduction Cytochrome P450 (CYPs) - important element of oxidative Cytochrome P450 (CYPs) - important element of oxidative metabolism metabolism Heme-containing Heme -containing monooxygenase monooxygenase proteins proteins Responsible for oxidative metabolism of different endogenous and Responsible for oxidative metabolism of different endogenous and exogenous compounds exogenous compounds 7 of the 57 known human P450s are responsible for more than 90% 7 of the 57 known human P450s are responsible for more than 90% of the metabolism of all pharmaceuticals in current use of the metabolism of all pharmaceuticals in current use Some of them display polymorphisms which can result in the poor Some of them display polymorphisms which can result in the poor metabolism pf drugs metabolism pf drugs Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Introduction Introduction They are found in almost all eukaryotes, prokaryotes, plants and They are found in almost all eukaryotes, prokaryotes, plants and even in hyperthermophilic archea. even in hyperthermophilic archea. ~1000 genes/enzymes are avilable ~1000 genes/enzymes are avilable till date till date Divided into 70 different families. Divided into 70 different families. Classifications of families - based on nucleotide sequence similarity Classifications of families - based on nucleotide sequence similarity Enzymes with less than ~40% sequence identity are placed in Enzymes with less than ~40% sequence identity are placed in different gene families different gene families Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Cytochrome P450 --Structures Cytochrome P450 --Structures CYP enzymes are membrane bound proteins, So difficult to crystallize. CYP enzymes are membrane bound proteins, So difficult to crystallize. First crystal structure of P450 was solved in the year 1985, it was from First crystal structure of P450 was solved in the year 1985, it was from pseudomonas putia pseudomonas putia with a resolution of 2.6 with a resolution of 2.6 Å Å. . After 2 decades, in 2000 the first mammalian P450 structure was After 2 decades, in 2000 the first mammalian P450 structure was determined. determined. CYP proteins have large binding pockets accommodating all exogenous CYP proteins have large binding pockets accommodating all exogenous and endogenous substances. and endogenous substances. CYPs have 6 Substrate Binding Sites (SRS), enabling CYPs to assimilate a CYPs have 6 Substrate Binding Sites (SRS), enabling CYPs to assimilate a large number of compounds. large number of compounds. Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Cytochrome P450 --Structures P450 --Structures Cytochrome Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Cytochrome P450 -- Structures Cytochrome P450 -- Structures PDB ID Date Resolution Source 1nr6 12/08/2003 2.1 Oryctolagus cuniculus 1po5 7/10/2003 1.6 Oryctolagus cuniculus 1tqn 27/07/2004 2.05 Homo sapiens 1jpz 9/11/2001 1.65 Synthetic Pseudomonas putida 1izo 18/03/2003 2.1 Bacillus subtillis 1n97 25/02/2003 1.8 Thermus thermophilus 1cpt 26/11/1993 2.3 Pseudomonas Sp 1jfb 20/12/2001 1 Fusarium oxysporum 1odo 2/01/2004 1.85 Streptomyces coelictor 1eup 28/04/2000 2.1 Saccaropolyspora erythrea 1q5d 23/10/2003 1.93 Polyangium cellulosum 1h5z 3/10/2003 2.05 Mycobacterium tuberculosis 1io7 28/02/2001 1.5 Sulfolbus sulfataricus 1lfk 11/12/2202 1.7 Streptomyces coelictor 1dz4 20/07/2000 1.6 Pseudomonas putida Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Method - Overview Method - Overview Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Method – – Structural Analysis Structural Analysis Method 15 structures were compared pair-wise using FATCAT (Flexible structure 15 structures were compared pair-wise using FATCAT (Flexible structure Alignment by Chaining Aligned fragment pairs allowing Twists) Alignment by Chaining Aligned fragment pairs allowing Twists) Alignment of one-to-one carbon atoms of each pair of protein structures. Alignment of one-to-one carbon atoms of each pair of protein structures. From FATCAT analysis From FATCAT analysis – – FATCAT scores (Structural Similarity) FATCAT scores (Structural Similarity) – Sequence Identity and Similarity Sequence Identity and Similarity – Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Methods – – Phylogenetic Analysis Phylogenetic Analysis Methods Sequences were aligned using Multiple Sequence Alignment. Sequences were aligned using Multiple Sequence Alignment. Aligned amino acids are subjected to tree building Aligned amino acids are subjected to tree building – PAUP* - Maximum Parsimony PAUP* - Maximum Parsimony – – – PHYLIP using Maximum Likelihood method with molecular clock and PHYLIP using Maximum Likelihood method with molecular clock and Jones-Taylor-Thornton model of amino acid change. Jones-Taylor-Thornton model of amino acid change. Evolutionary distances were calculated from the inferred trees. Evolutionary distances were calculated from the inferred trees. Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Results Results SIMILAR Vs IDENTICAL AMINO ACID 50 45 Rabbit Human with Rabbit 40 Human with IDENTICAL 35 30 25 20 Human with Archaea Human with Archaea 15 10 5 15 25 35 45 55 65 75 SIMILAR • The amino acid similarity between CYPs is approximately double he amino acid similarity between CYPs is approximately double the sequence identity the sequence identity • Property of the amino acid play an important role in structure- Property of the amino acid play an important role in structure- • function than its identity. function than its identity. Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Results Results FATCAT SCORE Vs E VOLUTIONARY DISTANCE Rabbit Human with Rabbit Human with 1500 1000 500 Human with Archaea Archaea Human with 0 0 1 2 3 4 5 6 7 DISTANCE DISTANCE The structures are similar in most aspects, except at some The structures are similar in most aspects, except at some positions, which are the functionality sites of these proteins. positions, which are the functionality sites of these proteins. In SRS 1,6,2 there is little conserved sequences and structures. In SRS 1,6,2 there is little conserved sequences and structures. In SRS 3-5 there is a considerable similarity in the protein In SRS 3-5 there is a considerable similarity in the protein structures. structures. Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
Observations Observations The Heme Heme binding are conserved and they exhibits a unique pattern. binding are conserved and they exhibits a unique pattern. The Significant conservation in SRS3-5. This conversation observed is in F/G Significant conservation in SRS3-5. This conversation observed is in F/G helix region which forms the major binding region. helix region which forms the major binding region. The catalytic residues of CYPs are present in the helix region of the CYP The catalytic residues of CYPs are present in the helix region of the CYP proteins. proteins. The tyrosine residue in the catalytic site plays an important role in the The tyrosine residue in the catalytic site plays an important role in the catalysis because the Iron ion oxidises the tyrosine side chain and produces catalysis because the Iron ion oxidises the tyrosine side chain and produces a tyrosyl tyrosyl free radical, which is essential for catalysis. free radical, which is essential for catalysis. a Gowri Shankar, University of Sydney. Gowri Shankar, University of Sydney.
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