Diversity in the Structure and Function of Amylase - PowerPoint Presentation

Slide1

Diversity in the Structure and Function of Amylase

Kim

Gernert

Emory University, Atlanta, GA

Vedham

Karpakakunjaram

Montgomery College, Rockville, MD

Slide2

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

Slide3

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

Slide4

Overview

Phylogenetic treeshttps://

sites.google.com/site/plasmodiumproblem/intro/about-phlyogenetic-trees

Blast

Protein sequence, amino acids

Protein structure, 3-D and secondary structural elements.

Slide5

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

Slide6

Question # 1

What is the phylogenetic relationship between representative organisms from the three domains, in terms of evolution of amylase?

Slide7

Study Organisms: a sample

Domain

Organism

Habitat/Lifestyle

Bacteria

E. coli

In

animal gut

Halothermothrix

orenii

Halothermophilic

Archaea

Pyrococcus

horishikii

HyperthermophilicP. woeseiEukaryaSaccharomycopsis fibuligeraUnicellularTenebrio molitorBeetle

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)

Slide8

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

Slide9

Tools

Multiple alignments CLUSTALW

construct

phylograms

in:

www.phylogeny.fr

Sequence alignments Blast

http://blast.ncbi.nlm.nih.gov/Blast.cgi

Slide10

Phylogeny based on Amylase

Slide11

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?

Slide12

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

Slide13

Sequence identity and

phylogeneticsby

Blast

Slide14

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

?

Slide15

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.

Slide16

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

Slide17

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?

Slide18

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

.

Slide19

Tools

Resources:

Use NCBI (

http://www.ncbi.nlm.nih.gov/

)

for comparing the identity and percent similarities in the sequences across organisms

Slide20

Tools

Resources:

Use Chimera (

www.cgl.ucsf.edu/chimera

) to visualize and compare the amylase structure in various organisms

Slide21

How does the structure of the active site visually compare across different organisms?

Discussion

Slide22

Multiple structure

overlay

Slide23

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?

Slide24

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.

Slide25

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)

Slide26

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

.