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/plasmodiumproblem/i
ntro/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 (1000s of known amylase

sequences)
17 molecular structures are presented (100s 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.