SEARCHGTr is a web based software for analyzing Glycosyltransferases (GTrs) involved in natural product biosynthesis. This software has been developed based on a comprehensive analysis of sequence/structural features of 102 GTrs from 52 natural product biosynthetic gene clusters and chemical structures of their donor/acceptor substrates. SEARCHGTr uses a knowledge-based approach wherein various predictions are carried out by comparing the query sequence with GTrs of known specificity.

Steps for analysis of query sequence

The query interface prompts the user to provide a short name for the query and the query sequence in FASTA format.
A link to an example query sequence is also provided.



On submission of the sequence, a page displaying a link to the result page appears. The results page provides each result in the form of separate blocks like:- FASTA, Related Glycosyltransferase Sequences (By Needleman and Wunsch -Global Alignmnet), Related Glycosyltransferase Sequences (By BLAST-Local Alignment), N- and C- domains, Substrate Binding Residues etc. Each block is explained individually:

The FASTA links to a pop up window displaying the query sequence in FASTA format.


 
 


 

As a first step in SEARCHGTr, the query sequence is compared with the GTrs in the GTrDB. The sequences are aligned with all the 102 entries in GTrDB using both local alignment program BLAST as well as Needleman and Wunsch global alignment program.
Using a pull down menu, the user can interactively view the list of GTr sequences homologous to the query and sort them as per their score/E-value, percentage identity, percentage similarity etc. Clicking on the button, shows the results in a tabular format.

As shown above for NW alignment, similar results are available for BLAST- Local alignment program.



Using the best matching GTr from GTrDB and its precomputed threading alignment with 1RRV, SEARCHGTr identifies the N- & C- terminal domains and linkers in the query sequence. These are then depicted in a pictorial format in a pop up window.
The numbers below the image of the domain specify its start and end positions on the query sequence. If there are parts of GTr,which are, overhangs on the N-terminal or C-terminal domains, they are shown as N-tail and C-tail respectively. The FASTA sequence of the domain or linker or tail can be viewed by clicking on the respective images.


The program also identifies putative Substrate Binding Residues (SBR) in the query sequence from the threading alignment of its best match with structural template. The user can choose either 1RRV or 1PN3 as the template for identifying the acceptor/donor binding residues. Clicking on the button, shows the results in a tabular format.




Additionally, SEARCHGTr provides an option for comparing the query sequence with the experimentally characterized GTrs in terms of their substrate (acceptor/donor) binding pocket residues alone. Here again, the user can choose 1RRV/1PN3 as the template for identification of SBR. The query SBR are pairwise compared with SBR of all GTrs in GTrDB and each position is scored using the BLOSUM62 matrix. The results are presented in a tabular format, which displays a sorted list of the best matching acceptor/donor-binding residues from GTrs in GTrDB along with their donor/acceptor specificities. In addition, the table displays the alignment of the acceptor/donor binding residues of each GTr with the query and its score. This allows one to one comparison of amino acids at each position of donor/acceptor binding pocket.



Furthermore, the program also has options for alignment of the query sequence with any specific GTr in GTrDB by selecting them based on the type of donor substrate, acceptor substrate or type of antibiotic.
Option 1 allows pairwise alignment (both global and local) of the query sequence with GTrs of any specific antibiotic.

In option 2, the program allows comparison of the query with GTrs which transfer any particular donor sugar to its acceptor. The figure below shows a typical result on selecting glucose as donor sugar. As can be seen, the program gives the list of GTrs which transfer glucose and their respective acceptors. It also shows the comparison of the DBR of each matching GTr and query and their equivalent scores. Links are provided to view the alignments of query and matching GTr.



In option 3, the program allows comparison of the query with GTrs which transfer any sugar to a particular class of acceptor molecules. This option provides results analgous to option2, where comparisons of acceptor binding residues are done, rather than donor binding residues.