A MORE CHALLENGING SEQUENCING EXERCISE

some more sequencing work, this one a little heavier than earlier 16s work.
(the figure numbers are off because this is the part 2 of a long paper...)

Protein Sequence: pro.50_orf_10
 (488 letters)
MHISSLPGKFGIGTFGKSAFEFVDFLAETKQTYWQILPLTTTSFGDSPYQSFSAIAGNTHFIDFELLVDDELLEAADLCDITFGTNPEAVDYAQLFQVRRPLLEKAVRAFVAEQENVCKLEAFETASSWLTDFAEFMALKEYFNNKALQDWDDETVIKRQEDSLNNYRELLAKKITYHKVCQYFFYQQWSALKTYANHKGIEIIGDMPIYVSADSVEVWTMPELFKVDSDKKPLFIAGVPADGFSEDGQLWGNPTYNWSAHEKSNFAWWIYRIQESFKLYDQLRIDHFKGFSDFWEIPAGDKTARNGHWASAPGIALFSAVREALGELPIIAENLGYIDEKAEQLLASTGFPGMKILEFGLFDITSQSIDLPHYYDRNCVAYTGTHDNEVVNGWYDNLSEEQVHFVNNYLHKHADESITKAMLRTIFASVCDTAILCIQDLLDKDGKSRMNMPNTIGGNWQWRMLDGELNQDHKDYLIYLTDLYGRAR

NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to search pro.50_orf_10 



Figure 27: NCBI BLAST results for pro.50_orf_10
The protein sequence was searched in NCBI BLAST and was identified as PRK14508, part of the Glyco_hydro_77 superfamily. 


Figure 28: NCBI BLAST color key for alignment scores for pro.50_orf_10
The color key for alignment scores is a graphical representation of E values for the matching sequences.  The shortened line is a partial match to a sequence, while the full length lines have the best E values; are more complete matches. 

Figure 29: NCBI BLAST partial match for pro.50_orf_10
The image above is an example of a partial match.  The E value is 0 and it is a 99% identity (the mismatched amino acid can be seen in the first line, there is a threonine instead of an isoleucine) however the sequence begins matching from the 167th amino acid and matches until the end (letter 488).  A better match would be a more complete sequence match as follows:


Figure 30: NCBI BLAST top match for pro.50_orf_10
The top match for pro.50_orf_10 is 4-alpha-glucanotransferase from organism Streptococcus pyogenes M1 GAS.  There is 100% identity and no gaps.  The E value is 0 and the match is from the first amino acid through the last (letter 488).    




Figure 31: NCBI BLAST Gene Details of 4-alpha-glucanotransferase
Going to the gene details of the top match, a list of related proteins is shown.  The protein from our match is fourth from the top of the list, from S. pyogenes M1 GAS.  the gene is called “malM” and is known as SPy_1292 in this particular organism. 



Figure 32: NCBI BLAST Gene Details of malM SPy_1292

Figure 33: NCBI BLAST Pathway information for malM SPy_1292
Selecting malM Spy_1292 more details are obtained on 4-alpha-glucanotransferase.  The gene is protein coding, is involved in starch and sucrose metabolism, and may also be called amylomaltase.  Two of the pathways listed are specific to S. pyogenes M1 GAS and a preview image is provided:



Figure 34: malM SPy_1292 pathway involvement in S. pyogenes M1 GAS

Figure 35: malM SPy_1292 starch and sucrose metabolism pathway involvement in S. pyogenes M1 GAS

KEGG database ( http://www.kegg.jp ) was searched for Spy_1292 to confirm NCBI BLAST pathway results. 


Figure 36: KEGG database results for SPy_1292
Both results from KEGG database search are for the correct organism, S. pyogenes M1 GAS but the first match is for the correct protein.  Selecting this provides detailed information:

Figure 37: KEGG database results for Spy_1292: malM; 4-alpha-glucanotransferase; K00705 4-alpha-glucanotransferase
KEGG database search confirms NCBI BLAST results.  Gene malM is also known as 4-alpha-glucotransferase from Streptococcus pyogenes serotype M1.  It is involved in two pathways for this organism, carbohydrate metabolism and starch and sucrose metabolism.  It is in the superfamily glyco_hydro_77 as identified by the conserved motif.


Figure 38: Metabolic pathways involved in S. pyogenes M1 GAS

The overview metabolic pathway for S. pyogenes M1 GAS shows applicable pathways in colors (all other are grayed out) and the specific gene searched is indicated in red.  KEGG shows malM as part of the sucrose and starch metabolism pathway. 
The sucrose and starch pathway for S. pyogenes M1 GAS is shown in detail:

Figure 39: KEGG database starch and sucrose metabolism pathway for S. pyogenes M1 GAS
malM is indicated in red (2.4.1.25) and is involved in metabolizing Maltose to alpha-D-Glucose.


To confirm NCBI BLAST results pro.50_orf_10 was searched in interpro database (https://www.ebi.ac.uk/interpro/ )

Figure 40: Interpro results for pro.50_orf_10 search
Interpro confirmed NCBI BLAST results and provided more information about the superfamily in which malM belongs to and its biological function (4-alpha-glucotransferase activity in carbohydrate metabolic process). Glycol_hydro_77 superfamily is Glycoside hydrolase family 77. 


Figure 41: Details of glyco_hydro_77 from Interpro search of pro.50_orf_10
Details of glycol_hydro_77 confirm KEGG results; the family is involved in metabolism of alpha-D-Glucan.


Pfam database (http://pfam.xfam.org) is searched for pro.50_orf_10

Figure 42: Pfam results for pro.50_orf_10
Pfam confirms Interpro results, showing 4-alpha-glucanotransferase in the glycol_hydro_77 family.  The detailed information also confirms the description by Interpro.


Figure 43: Detailed results of Pfam search for pro.50_orf_10
2557 different organisms use the Glyco_hydro_77 family. 

Figure 44: Sunburst diagram of Glyco_hydro_77 family distribution among domains
A sunburst diagram shows the distribution is highest among bacteria although other domains contain this family.  Streptococcus pneumoniae has a higher frequency of the family (170 species) among Streptococcus species; S. pyogenes distribution is shown in the alt-text box (15 species).
Pro.50_orf_10 was searched in SMART database but did not have any results.  Instead, pro.50_orf_10 was searched in Uniprot BLAST (http://www.uniprot.org ) to obtain an accession number. 

Figure 45: Uniprot BLAST result for pro.50_orf_10
Uniprot BLAST search resulted in accession number Q99ZC2.
The accession number obtained was searched in SMART database but had no results, possibly because the protein is an enzyme and is only a predicted structure (SMART specializes in evolutionary conserved, regulatory domains: http://smart.embl-heidelberg.de/help/FAQ.shtml )
Instead, STRING database was accessed directly (http://string-db.org/newstring_cgi/show_input_page.pl?UserId=fhcW4mKEyaBx&sessionId=6yzve2qn3GK1 ) to search for malM in S. pyogenes M1 GAS.

Figure 46: STRING database results for malM in S. pyogenes M1 GAS
malM is confirmed as 4-alpha-glucotransferase in STRING database and has functional partners glycogen phophorylase (glgP) and cyclomaltodextrinase (amyB). 


SUMMARY

Two unknown amino acid sequences were researched across five databases... The second sequence, pro.50_orf_10, comes from Streptococcus pyogenes M1 GAS and is in the family Glycoside Hydrolase Family 77.  The gene is malM also known as 4-alpha-glcotransferase and is involved in the metabolism of starch and sucrose. 


and now we party because it's over!



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