Transcript source file

Enzymatic Function Module
(KEGG, MetaCyc, and EC Numbers)
Provide contextual information
about your gene as component
of pathway or cellular structure
Provides information about
the class to which an enzyme
belongs:
 oxidoreductases
 transferases
 hydrolases
 lysases
 isomerases
 ligases
Kyoto Encyclopedia of Genes and Genomes
- KEGG is a collection of biological information compiled from
published material  curated database.
- Includes information on genes, proteins, metabolic pathways,
molecular interactions, and biochemical reactions associated with
specific organisms
- Provides a relationship (map) for how these components are
organized in a cellular structure or reaction pathway.
The Good: Information is reliable!
The Bad: Information is not available for many organisms.
Recall: Many used Rhodopirellula baltica as the reference genome
for our initial gene hunt (Why? It is in the same family as P. limnophilus)
Recall: You have already entered
the information from the KEGG
database into your team notebook.
If not, you should do it now.
NOTE: Refer to the week 2
student presentation for details
Recording results in your individual notebook
Refers not to the team pathway,
but to your individual gene.
This information may be found
on the Gene Detail page or
determined directly from the
KEGG pathway database.
Go to Gene Detail page for your gene
Scroll down to Pathway Information
for your gene
Notice your gene
may be involved in
KEGG pathways
other than the one
you are annotating
with your team.
Why? Your gene may
function in more than
one pathway in the cell.
• Sequentially click on the link for each pathway.
 Note the EC number if your ORF is an enzyme in a biochemical pathway
or the gene name if your ORF is an protein component of a cellular complex
so you can locate your ORF on the KEGG map.
• SAVE each KEGG map in PNG format then upload to your notebook.
OID 2500609751
Note the color scheme for KEGG maps differ
from that obtained directly from database
EC 6.3.1.2
OID 2500609751
KEGG Pathways for EC 6.3.1.2
Recording results in your individual notebook
1- Modify heading
2- Insert KEGG map
3- Include comment
Recording results in your individual notebook
4- Repeat for all
KEGG maps
Scroll
down
What if no KEGG pathways listed on Gene Detail page?
1- Try pathway text search
Enter part or
all of the gene
product name
“Click”
Pathway text search results
KEGG pathway names
“Click”
KEGG
maps
Pathway text search results
“Click” for
larger image
Inspect map
for EC number
or gene name
corresponding
to your gene
What if no KEGG pathways listed on Gene Detail page?
2- Try KEGG2 search
“Click”
Enter part or
all of the gene
product name
“Click”
KEGG2 Search Results
“Click”
KEGG2 Search Results
“Click”
Inspect map
for EC number
or gene name
corresponding
to your gene
MetaCyc
- Database of nonredundant, experimentally
elucidated metabolic pathways  curated database
- Goal is to catalog the universe of metabolism
by storing a representative sample of all pathways
that have been experimentally elucidated
Caspi et al. (2008)
NAR 36: D623-631.
Click the link in your notebook
Note that this database
only works properly in
Firefox browser.
Select the database to be used for your search
“click”
Select Pirellula sp.
(It is in the same family
as P. limnophilus)
“click”
Perform a BLAST search against Pirellula sp. genome
Reveal drop-down menu
under Search function tab
Note the database
name has changed
“click”
Note the BLAST
search functions
are on the
BioCyc site
Make sure you perform
a protein BLAST search
Copy/paste the amino acid
sequence for your gene in
FASTA format into query box
Change E-value to 0.01
Leave other settings
as default
“click”
Inspect your BLAST search results for significant hits
Look at your top hit:
Alignment corresponds
to only 73 / 470 = 16%
- check for a positive bit score
- confirm that the E-value
is less than 10-3
- make sure identity 30 to 35%
and that the alignment covers
at least 50% of query sequence
If the search does not
produce significant hits,
or if the top hits have
fairly low score & high
E-value, try searching a
different database:
E. coli K-12 substr. MG1655
Note the BLAST
search functions
for E. coli are on
the EcoCyc site
“click”
Inspect your BLAST search results for significant hits
Look at your top hit:
Alignment corresponds
to only 470 / 470 = 100%
- check for a positive bit score
- confirm that the E-value
is less than 10-3
- make sure identity 30 to 35%
and that the alignment covers
at least 50% of query sequence
Click on link for gene
name for top hit that
meets above criteria;
otherwise write
“No significant hits” in
your lab notebook
The results will look something like this..
- Compare the name of the search hit
to the name of the protein on your
Gene Detail page in img/edu
- For example: “glutamine synthetase”
to “L-glutamine synthetase”
Examine the “Gene-Reaction Schematic”
which you will copy into your lab notebook
The Gene-Reaction Schematic depicts the relationships
among a set of genes, enzymes, and reactions:
NOTE: A line indicates
a relationship
between two objects.
For example, a line
from a gene to a circle
indicates that gene
encodes that product)
For more information
about the schematic,
click on the ? symbol
The circle represents
a polypeptide
or protein product
The box on the left
represents the enzyme
catalyzing the reaction
The letters on the right
represent the gene
Scrolling over each
of the symbols will
show a text box
with additional
information
Modify your notebook as follows:
Enter the following information
into your lab notebook
Gene information:
Name & EC number
To SAVE the reaction schematic:
•
•
•
Gene-Reaction
Schematic
Scroll over a LINE (not circle,
box, or surrounding space)
Right-click to bring up window
with option to “Save Image As”
SAVE in .gif format to upload
to the notebook.
Recording results in your notebook
EC Number
Enzyme Commission (EC) Number is a series
of 4 numbers describing enzymatic function:
1: Indicates which of the 6 classes the enzyme belongs:
oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase
2 and 3: Depends on the enzyme class
For example, in oxidoreductases: 2 describes the substrate,|
and 3 describes the acceptor)
4: Gives the specific enzyme activity
How do you determine the EC number?
First ask yourself: Is my gene an enzyme or a structural gene?
Only enzymes have EC numbers. . .
Four options to try:
1- Inspect the img/edu Gene Detail page
2- Look at KEGG results
3- Examine the MetaCyc results
4- Search the Expasy database
We will go through each option.
All should agree on the EC number
if assigned accurately by gene caller.
Inspect the img/edu Gene Detail page
Scroll
down
“click”
EC number
EC name
Look at KEGG results
“click”
EC number
EC name
Examine the MetaCyc results
EC name
EC number
Search the Expasy database
Scenario: By gene name/description, you know you have
an enzyme to annotate but are not sure what MetaCyc or
KEGG pathway it belongs to – what do you do?
1- Go to Expasy at http://www.expasy.org/enzyme/
NOTE: You must access the website
via the above URL. The link in the
notebook takes you to a different part
of the Expasy site – do not use it.
2- Enter the name or description of your gene then press
[Search] button
3- Results should produce a list of possible genes with
more detailed descriptions & associated EC numbers
EC number
4- Depending on the description for your gene in P. limnophilus
or other evidence you've accumulated through the quarter
(see BLAST results for example), you may be able to narrow
down which one it is . . . Put some thought into it and see what
you can come up with. . .
click
. . . If you click on the EC number for a candidate, it will
take you to a page with links to KEGG and MetaCyc.
5- Click the link to KEGG to see a detailed results page
click
NOTE: There also are links to other databases,
including PubMed (MEDLINE) and MetaCyc.
6- On detailed results page, click [Show all] for
KEGG reaction to see schematic
click
7- Inspect the reaction schematic(s). Is the equation
consistent with proposed enzyme function?
Click here for names of
compounds in the reaction
Click to obtain
high-resolution
image
EC name
EC number
8- Optional: You may add high-resolution image
to your notebook. SAVE as .gif file and
upload to your notebook.
Recording results in your notebook
REMEMBER: Not all genes will have an EC
number. Only genes with enzyme function are
assigned an EC number. If your gene ends with
‘ase’, it could be an enzyme. Remember,
enzymes have catalytic function.
Module tasks complete
Are you keeping up with your annotations?
The 2nd of 3 imgACT notebook checks
will occur at the end of this week.