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Seq205-01

    Seq 205 Annotation Main

    Amino acid sequence for Seq 205-1 was analyzed with InterProScan to reveal a glycoside hydrolase family 28 domain,  parallel beta-helix repeat,  pectin lyase fold/virulence factor, and  pectin lyase as shown below:

     

    interpro 205-01.jpg

     

    Glycoside hyrdolase, family 28

    The glycosyl hydrolase family 28 contains important cell wall metabolism enzymes such as polygalacturnose and rhamnogalacturonase.  These enzymes hydrolyse the glycosidic bonds between carbohydrates or a carbohydrate and a non-carbohydrate moiety.  Other members of the glycoside hydrolase family 28 include Rhamnogalacturonan is one of the fundamental components of pectin of cell walls.  Two forms of Rhamnogalacturonan have been identified: RG I and RG II.  The highly conserved RG II is characterized by its diversity of sugars and linage structures such as apiose, aceric acid, 2-O-methyl fucose, 2-O-methyl xylose, 3-deoxy-D-manno-2-octulosonic acid, and 3-deoxy-D-lyxo-2-heptulosaric acid.  RG I is characterized by its rod-like heteropolymer of repeating (1-->2) alpha-L-rhamnose-(1-->4) alpha-D-galacturonic acid disaccharide units and is digested by polygalacturonase.  Polygalacturonase is involved in two pathways, pentose and glucuronate interconversions and starch and sucrose metabolism, as listed below in the KEGG diagrams (highlighted in red).

    polygalact 1.png

    polygalact 2.png

    Seq

    ClustalW2 alignment with BLASTn of putative polygalacturonase. KEGG BLAST of "zma-FGENESH" nucleotide base sequence against these plant species revealed top matched genes. Five plant species were analyzed: zma= Z. mays, ath= A. thaliana, rcu= R. communis, vvi= V. vinifera, and osa= O. sativa japonica. "zma-FGENESH" is the sequence derived from the FGENESH analysis and "zma-KEGG" is the best BLASTed result analyzed through KEGG's BLAST tool.

    polygalt BLASTn.jpg

     

    ClustalW2 alignment with BLASTx of polygalacturonase. KEGG BLAST of "zma-FGENESH" AA sequence against these plant species revealed top matched genes. Five plant species were analyzed: zma= Z. mays, ath= A. thaliana, rcu= R. communis, vvi= V. vinifera, and osa= O. sativa japonica. "zma-FGENESH" is the sequence derived from the FGENESH analysis and "zma-KEGG" is the best BLASTed result analyzed through KEGG's BLAST tool.

    polgalt BLASTx.jpg

     

    MEGA 4.0 Neighbor-Joining, 1000 replicate bootstrap test of phylogeny. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. KEGG BLASTn of "zma-FGENESH"  sequence against these plant species revealed top matched genes. Five plant species were analyzed: zma= Z. mays, ath= A. thaliana, rcu= R. communis, vvi= V. vinifera, and osa= O. sativa japonica. "zma-FGENESH" is the sequence derived from the FGENESH analysis and "zma-KEGG" is the best BLASTed result analyzed through KEGG's BLAST tool.

    polygalt NJ.jpg

     

    It appears that Z. mays polygalacturonase is more similar to O. sativa japonica in whole nucleotide base and amino acid sequences. Both the BLASTn and BLASTx show a high much higher score with zma and osa than with any other plant species shown. The phylogenetic tree analysis concurs that indeed osa is more similar to zma than the other three plant species. However, in future work it would be interesting to take the sequences coding for the particular structures in the protein and compare those amongst plant species. Please note, that zma's BLAST result with vvi is a hypothetical protein, and needs much more annotation and bioinformatic analysis.

    Tertiary Structure Prediction

    Putative protein structures were determined by utilizing the protein modeling service I-TASSER (Roy et al., 2010; Zhang, 2008; Zhang, 2009).  I-TASSER predicted 5 top protein structures as ranked by the C-score.  The C-score is a confidence score used by I-TASSER to estimate the quiality of the predicted models.  C-scores typically fall in the range of -5.0 to 2.0.  Higher scores correlate with a better model.  More information on C-scores can be found here.  Comparison of the C-scores for each model are located below.  These putative proteins were loaded into Pymol and edited to highlight the main structural components such as the alpha helices (teal) and beta sheets (red).  Hydrogens and side chains were masked to produce a clearer image of the protein. Model 1 is shown as Image 1.  Other putative models are located in Supplemental Data, Images 1 to 5.

    205-01 C-Score Comparisons

    Model C-Score
    205-01 Model 1 -0.34
    205-01 Model 2 -1.07
    205-01 Model 3 -0.89
    205-01 Model 4 -2.58
    205-01 Model 5 -1.02

     

       

    Image 1: 205-01 Model 1

    205-01 model1.png

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