Enzymatic Function Module(KEGG, MetaCyc, and EC Numbers)

Provide contextual informationabout your gene as componentof pathway or cellular structure

Provides information aboutthe class to which an enzymebelongs:

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 genomefor our initial gene hunt (Why? It is in the same family as P. limnophilus)

Recall: You have already enteredthe information from the KEGGdatabase into your team notebook.If not, you should do it now.

NOTE: Refer to the week 2student presentation for details

Refers not to the team pathway,but to your individual gene.

This information may be foundon the Gene Detail page ordetermined directly from theKEGG pathway database.

Recording results in your individual notebook

Go to Gene Detail page for your gene

Scroll down to Pathway Information for your gene

OID 2500609751

Notice your genemay be involved inKEGG pathwaysother than the oneyou are annotatingwith your team.

Why? Your gene mayfunction in more thanone 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

KEGG Pathways for EC 6.3.1.2

1- Modify heading

2- Insert KEGG map

3- Include comment

Recording results in your individual notebook

4- Repeat for all KEGG maps

Scrolldown

Recording results in your individual notebook

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

KEGG maps

“Click”

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

MetaCycMetaCyc

- 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 databaseonly works properly inFirefox browser.

Select the database to be used for your search

“click”

Select Pirellula sp.(It is in the same familyas P. limnophilus)

“click”

Perform a BLAST search against Pirellula sp. genome

Note the databasename has changed

Reveal drop-down menuunder Search function tab

“click”

Note the BLASTsearch functions

are on the BioCyc site

Make sure you performa protein BLAST search

Copy/paste the amino acidsequence for your gene inFASTA format into query box

Change E-value to 0.01

Leave other settingsas default

“click”

Look at your top hit: - 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

Inspect your BLAST search results for significant hits

If the search does notproduce significant hits,or if the top hits havefairly low score & highE-value, try searching adifferent database:E. coli K-12 substr. MG1655

Alignment correspondsto only 73 / 470 = 16%

Note the BLASTsearch functionsfor E. coli are onthe EcoCyc site

“click”

Look at your top hit: - 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

Inspect your BLAST search results for significant hits

Alignment correspondsto only 470 / 470 = 100%

Click on link for genename for top hit thatmeets above criteria;otherwise write“No significant hits” inyour 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 relationshipsamong a set of genes, enzymes, and reactions:

NOTE: A line indicates a relationshipbetween two objects. For example, a line from a gene to a circle indicates that gene encodes that product)

The letters on the rightrepresent the gene

The box on the left represents the enzymecatalyzing the reaction

The circle representsa polypeptide

or protein product

Scrolling over each of the symbols will

show a text box with additional

information

For more informationabout the schematic,click on the ? symbol

Modify your notebook as follows:

Enter the following informationinto your lab notebook

Gene information:Name & EC number

Gene-ReactionSchematic

To SAVE the 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 NumberEC Number

Enzyme Commission (EC) Number is a seriesof 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?

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.

First ask yourself: Is my gene an enzyme or a structural gene?Only enzymes have EC numbers. . .

Inspect the img/edu Gene Detail page

Scrolldown

“click”

EC name

EC number

Look at KEGG results

EC name

EC number

“click”

Examine the MetaCyc results

EC name

EC number

Scenario:Scenario: By gene name/description, you know you havean enzyme to annotate but are not sure what MetaCyc orKEGG pathway it belongs to – what do you do?

Search the Expasy database

1- Go to Expasy at http://www.expasy.org/enzyme/

NOTE: You must access the websitevia the above URL. The link in the

notebook takes you to a different partof 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. limnophilusor other evidence you've accumulated through the quarter(see BLAST results for example), you may be able to narrowdown which one it is . . . Put some thought into it and see whatyou can come up with. . .

. . . If you click on the EC number for a candidate, it willtake you to a page with links to KEGG and MetaCyc.

click

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 to obtainhigh-resolutionimage

EC number

Click here for names ofcompounds in the reaction

EC name

8- Optional: You may add high-resolution image to your notebook. SAVE as .gif file and upload to 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.

Recording results in your notebook

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.