Diversity in the Structure and Function of Amylase Kim Gernert Emory University, Atlanta, GA Vedham Karpakakunjaram Montgomery College, Rockville, MD Target Audience • Students enrolled in Principles of Biology I (BI 107) and II (BI 108) • Human salivary amylase: used in one of our lab modules, so students are familiar with this enzyme and its function Background • Alpha amylase: http://www.rcsb.org/pdb/101/motm.do?momID=74 • Alpha-amylase: begins starch breakdown – Starch chains broken into two or three glucose units – Most organisms synthesize amylase Overview • Phylogenetic trees • https://sites.google.com/site/plasmodiumpr oblem/intro/about-phlyogenetic-trees • Blast • Protein sequence, amino acids • Protein structure, 3-D and secondary structural elements. Big Picture: Concepts • Evolutionary relationships between organisms in a molecular scale • Correlation between Structure and Function – Focus: modifications, if any, in amylase across organisms with unique lifestyles Question # 1 • What is the phylogenetic relationship between representative organisms from the three domains, in terms of evolution of amylase? Study Organisms: a sample Domain Organism Habitat/Lifestyle E. coli In animal gut Halothermothrix orenii Halothermophilic Bacteria Pyrococcus horishikii Archaea Hyperthermophilic P. woesei Saccharomycopsis fibuligera Unicellular Tenebrio molitor Beetle Eukarya 6 out of 20 sequences archived for the study (1000’s of known amylase sequences) 17 molecular structures are presented (100’s of solved structures) Data • The European Bioinformatics Institute sequences (http://www.ebi.ac.uk/) • Protein Data Bank Molecular Structures (http://www.rcsb.org/pdb/home/home.do) >Aspergillus oryzae 2gvy ATPADWRSQSIYFLLTDRFARTDGSTTATCNTADQKYCGGTWQGIIDKLDYIQGMGFTAI WITPVTAQLPQTTAYGDAYHGYWQQDIYSLNENYGTADDLKALSSALHERGMYLMVDVVA NHMGYDGAGSSVDYSVFKPFSSQDYFHPFCFIQNYEDQTQVEDCWLGDNTVSLPDLDTTK DVVKNEWYDWVGSLVSNYSIDGLRIDTVKHVQKDFWPGYNKAAGVYCIGEVLDGDPAYTC PYQNVMDGVLNYPIYYPLLNAFKSTSGSMDDLYNMINTVKSDCPDSTLLGTFVENHDNPR FASYTNDIALAKNVAAFIILNDGIPIIYAGQEQHYAGGNDPANREATWLSGYPTDSELYK LIASANAIRNYAISKDTGFVTYKNWPIYKDDTTIAMRKGTDGSQIVTILSNKGASGDSYT LSLSGAGYTAGQQLTEVIGCTTVTVGSDGNVPVPMAGGLPRVLYPTEKLAGSKICSSS Tools • Multiple alignments CLUSTALW • construct phylograms in: www.phylogeny.fr • Sequence alignments Blast • http://blast.ncbi.nlm.nih.gov/Blast.cgi Phylogeny based on Amylase Discussion • Give a general set of observations on the tree. • What clusters with the human sequences? • Identify mono-phyletic groups within the tree. • Why possibly the archaean species are isolated in the tree? Question 2 • What is the percent similarity in structure of amylase based on the phylogenetic tree? • How does the sequence identity of the sequences match the clustering in the phylogenetic tree? • Blast for percent sequence identity and percent sequence similarity. • This will help students to quantitatively connect the information from phylogenetic tree to secondary structure/sequence Sequence identity and phylogenetics by Blast Question # 3 • Are the amino acid sequences (hence the structure) different across organisms? • Where are the conserved regions in the molecular structure? • Do they relate to the secondary structural elements? Conserved Sequences Regions of the alignment that are highly conserved are highlighted in green and blue. The same sequences are colored on the 3-dimensional structure in Chimera. Tools • Use Chimera (www.cgl.ucsf.edu/chimera) to visualize and compare the amylase structure in various organisms 05-pdbs-061313-006.py Structure file including human, porcine, beetle and bacterium amylase Discussion • What sections of the structure are colored green? • What sections of the structure are colored blue? • Why are the sequence of these regions conserved? • What other regions do you think will be conserved? Question # 4 • Note that 4 or more conserved residues in a row highlight a critical region of the protein structure. • The active site residues are in these regions. Tools • Resources: − Use NCBI (http://www.ncbi.nlm.nih.gov/) for comparing the identity and percent similarities in the sequences across organisms Tools • Resources: • Use Chimera (www.cgl.ucsf.edu/chimera) to visualize and compare the amylase structure in various organisms Discussion • How does the structure of the active site visually compare across different organisms? Multiple structure overlay Discussions • What are the critical active site residues? • Are they present in all of the structures from different species? • Which structures have glucose or another starch bound? • Do the different species bind the starch differently? Future plans • Study the binding of other molecules in the active site including inhibitors. • Study substrate analogs. • Role of mutations in modifying the structure and function of amylase. Known mutations • Mutations, structural and functional effects. 1xgz MUTANT N298s 1nm9 MUTANT W58A subsite 2. Critical for enzyme activity. 1q4n MUTANT F256W salivary 1kgu pancreatic MUTANT R377A (probing role of chloride ion). 1kgw pancreatic MUTANT R337Q 1kgx pancreatic MUTANT R195Q (probing role of chloride ion). Hordeum vulgare (barley) 2xfr (2010, 0.97A) 2xff (2010, 1.31) H395a, (y105a, y308a), d180a (inactive) Glycine max (soybean) 1v3h, 1v3i soybean, E186, E380 (resolution 1.6 A) 1q6c, complex with maltose, M51T, E178Y, N340T (increased pH optimum) Bacillus cereus 1vem (2005, 1.85) Y164e, (t47m, Y164e, t328n) Bibliography • http://www.ebi.ac.uk/ • http://www.rcsb.org/pdb • General textbook • • • 1hny Protein Sci. 1995 Sep;4(9):1730-42. The structure of human pancreatic alpha-amylase at 1.8 A resolution and comparisons with related enzymes. Brayer GD, Luo Y, Withers SG. 1smd Acta Crystallogr D Biol Crystallogr. 1996 May 1;52(Pt 3):435-46. Structure of human salivary alpha-amylase at 1.6 A resolution: implications for its role in the oral cavity. Ramasubbu N, Paloth V, Luo Y, Brayer GD, Levine MJ.