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.