Annotation Presentation Week 7 Enzymatic Function Module (KEGG, MetaCyc, and EC Numbers) 1

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 2

Kyoto Encyclopedia of Genes and Genomes KEGG 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)

NOTE: Refer to the week 2 student presentation for details 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 4

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 5

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 6

Note the color scheme for KEGG maps differ from that obtained directly from database OID 2500609751 7

KEGG Pathways for EC 6.3.1.2 8

Recording results in your individual notebook 1- Modify heading 2- Insert KEGG map 3- Include comment 9

Recording results in your individual notebook 4- Repeat for all KEGG maps Scroll down 10

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” 11

Pathway text search results KEGG pathway names KEGG maps “Click” 12

Pathway text search results “Click” for larger image Inspect map for EC number or gene name corresponding to your gene 13

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” 14

KEGG2 Search Results “Click” 15

Inspect map for EC number or gene name corresponding to your gene KEGG2 Search Results “Click” Inspect map for EC number or gene name corresponding to your gene 16

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. 17

Click the link in your notebook Note that this database only works properly in Firefox browser. 18

Select the database to be used for your search “click” Select Pirellula sp. (It is in the same family as P. limnophilus) “click” 19

Perform a BLAST search against Pirellula sp. genome Reveal drop-down menu under Search function tab Note the database name has changed “click” 20

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” 21

Inspect your BLAST search results for significant hits 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 Alignment corresponds to only 73 / 470 = 16% 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 22

Note the BLAST search functions for E. coli are on the EcoCyc site “click” 23

Inspect your BLAST search results for significant hits 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 Alignment corresponds to only 470 / 470 = 100% Click on link for gene name for top hit that meets above criteria; otherwise write “No significant hits” in your lab notebook 24

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” 25

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: For more information about the schematic, click on the ? symbol 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) 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 26

Modify your notebook as follows: 27

Enter the following information into your lab notebook Gene information: Name & EC number 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. Gene-Reaction Schematic 28

Recording results in your notebook 29

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 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 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

7- Inspect the reaction schematic(s) 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 number EC name

8- Optional: You may add high-resolution image to your notebook 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.