Tools :

1. Diversity in the Structure and Function of Amylase • Kim Gernert and VedhamKarpakakunjaram Homo_sapieALGKDYVRSKIAEYMNHLID-IGVAGFRIDASKHMWPGDIK--------------AILDK n3VM5_Meda ALEKDYVRGKVADFMNKLID-MGVAGFRVDACKHMWPGDLD--------------NVYRR Tenebrio_mNQGSDYVRGVLIDYMNHMID-LGVAGFRVDAAKHMSPGDLS--------------VIFSG sp|P25718| GTFHGGDLRGLTNKLDYLQQ-LGVNALWISAPFEQIHGWVGGGTKGDFPHYAYHGYYTQD SaccharomyRTEDSDVASVFNSWVKDFVGNYSIDGLRIDSAKHVDQGFFP-------------DFVSAS Oryza_satiDHLNKRVQRELIGWLDWLKMDIGFDAWRLDFAKGYS----------------------AD PyrococcusTHELVYERGWLKEFFDRISSDDKINLMLYSEYLSKFRPKGLVYLPIASYFEMSEWSLPAR . .. : . .   Homo_sapieLHNLNSNW-FPEGSKPFIYQEVID----LGGEPIKSSDYFGNGRVTEFKYGAK—LGTVI n3VM5_Meda LNNLNTKW-FPGGSRPFIFQEVID----LGGEPITTGEYVGLGRVTEFKYGAR—LGELF Tenebrio_mLKNLNTDYGFADGARPFIYQEVID----LGGEAISKNEYTGFGCVLEFQFGVS—LGNAF sp|P25718| WTNLDANMGNEADLRTLVDSAHQRGIRILFDVVMNHTGYATLADMQEYQFGALYLSGDEV SaccharomyGVYSVGEVFQGDPAYTCPYQNYIP----------GVSNYPLYYPTTRFFKTTDSSSSELT Oryza_satiMAKIYIDATEPSFAVAEIWTSMAN----------GGDGKPNYDQNAHRQELVNWVDRVGG PyrococcusQAKLFFEFIKKLKELNLFEKYRIFVRG------GIWKNFLYKYPEGNYMHKRMLMLSKLL Target audience: Students enrolled in Principles of Biology I (BI 107) and II (BI 108) Background Alpha amylase: http://www.rcsb.org/pdb/101/motm.do?momID=74 Alpha-amylase begins the process of starch digestion. It takes starch chains and breaks them into smaller pieces with two or three glucose units. Human salivary amylase is used in one of our lab modules, so students are familiar with the enzyme and its function. Questions Given organisms from three domains with diverse lifestyles and study sequence differences and their effect on enzyme’s structure and function. Are the structures of amylase different across organisms? Relate the identity and percentage similarities in sequences based on clustering in the phylogenetic tree. Relate the conserved regions of the alignment to the secondary structure of amylase. Identify the active site and locate the critical residues and find their conservation of the sequences in the alignment. Study the binding of other molecules in the active site including inhibitors. How do mutations in amylase modify their structures? How do these modifications alter the enzyme’s function? • Tools: • Amino acid sequences (PDB, EBI) from many diverse organisms to be provided for students to select about 5-6 organisms representing the three domains. • Hyperthermophilic Archeae, parasitic and mutualistic bacteria, unicellular and multicellular eukaryotes. • Compare the sequences with alignments from CLUSTALW and construct phylograms in: www.phylogeny.fr • Use NCBI (http://www.ncbi.nlm.nih.gov/) for comparing the identity and percent similarities in the sequences across organisms in order to synthesize the information from phylogenetic trees. • Use Chimera (www.cgl.ucsf.edu/chimera) to visualize: • conserved and modified regions, especially around the active site. • the binding of substrate, inhibitors • to determine the nature of inhibition (competitive) • Provide sequences of mutations to be compared against normal sequences for identification of conserved and modified regions, and to predict modifications in the mutant enzyme’s function, if any.