Lipase Biocomputing Project Presented By: Angel, Jason, Jeff and Kelly Outline • Background Information • Structure • Phylogeny/Homology Overview • Lipase responsible for fat absorption in the small intestine • Its activity depends on presence of pancreatic colipase and bile salts • Lipase insufficiency can be managed by diet • Lipase malabsorption can be induced by Xenical weight loss Background Information • Lipases hydrolyze triacylglycerols, releasing one fatty acid at a time and forming diacylglycerols, monoacylglycerol then finally, glycerol. • Lipase is essential in most if not all living organism • Lipases also used by fungi and bacteria to facilitate nutrient absorption from the external medium Background Information • Pancreatic Lipase – Released into duodonem – Hydrolyzes triacylglycerols (TAG) into free fatty acids and monoglycerides – TAGs are emulsified within bile salt micelles Main enzyme responsible for fat absorption in small intestine Background Information • Pancreatic Colipase (PC) – Secreted as procolipase in a 1:1 ratio with PL – PC can bind to the bile salt micelle – PC necessary for PL function Structure of Lipase Overview • Has only one protein chain (Chain A) – 534 amino acids long – 12 β sheets – 25 ά helices • No DNA chains • Has two Disulfide bridges • Has six ligands Ligands 1. (1R)-Menthyl hexyl phosphonate group •Center of the protein •Connected to lots of different Amino Acids •Two Serines and two leucines 2. – 3. Calcium Ions 2. Calcium Ion A •Interacts with Asparagine, amino acid # 260 •Also with one hetero atom 2. – 3. Calcium Ions 3. Calcium Ion B •Interacts with Glycine, amino acid # 326 •Connected to multiple heteroatoms 4. N-Acetyl-D-Glucosamine (NAG) •Interacts with asparagine 222, lysine 180, asparagine 314, Valine 313, and tryptophan 221. 5. NAG group •Two NAG ligands interacting with each other at the same site. •Also interact with Tyrosine 299, aparagine 351, valine 352, glutamine 357, and glutamate 70 Active Site • Not referenced in Protein Explorer, so determined via conserved regions. Protein Family • Multiple possibilities – Carboxylesterase (E of 6e-91) – Lipolytic Enzyme (E of 2.8e-5) – Hydrolase (E of 0.19) • However, often classified as a Hydrolase by various databanks (such as GenBANK file in NCBI). Structural Motifs • Multiple phosphorylation sites • Contains much N-terminal glycine which have a covalent bond with myristate (a C-14 saturated fatty acid). Predictive Software Analysis Ά Helices Β Sheets Predicted 11 11 Actual 25 12 Generally predicted the locations of helices and sheets, but not with perfect accuracy. Transmembrane Properties • Lipase is not an integral membrane protein •No interesting relatives Phylogeny Homologs to 1LPM ‘lipase’ • Gram negative bacteria • Homo sapien carboxylesterase • Possum carboxylesterase • Domestic horse butyrylcholinesterase • Homo sapien acetylcholinesterase • Roundworm acetylcholinesterase E-Values E-values of Homologs e-value Monodelphis domestica: 7e-41 Xanthomonas 8e-43 Homo sapien acetlycholinesterase 7e-40 Homo sapien carboxylesterase 4e-35 Meloidogyne 1e-41 Equus caballus 1e-41 Multiple Sequence Alignment •More conserved residues •Not many conserved groups Phylogenetic Tree • Unrooted Values used to determine bootstrapping values Phylogenetic Tree •Calculated Bootstrapping 100% values 100% 100% 88.4% What's interesting? • Expected bootstrapping values closer to 100% • Actually 88.4% • Less Confidence REFERENCES • Svendsen A (2000). "Lipase protein engineering". Biochim Biophys Acta 1543 (2): 223–228. • Winkler FK, D'Arcy A, and W Hunziker (1990). "Structure of human pancreatic lipase". Nature 343 (6260): 771–774. Algorithms Used • Protein Explorer – Jmol – Consurf – Biology Workbench (PELE, TMAP, GREASE, TMHMM, BLASTP, PROSEARCH, CLUSTALW, CLUSTALTREE, DRAWTREE ).
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