Computational protocol: Identification of the Catalytic Triad of Family S46 Exopeptidases, Closely Related to Clan PA Endopeptidases

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[…] Using primers recognizing universally conserved regions, 16S ribosomal gene fragments were amplified from Pseudomonas sp. strain WO24 genomic DNA, cloned and sequenced. This provided the information to determine the complete 1523 nt sequence of its 16S rRNA gene. Phylogenetic tree analysis of 16S rRNA sequences was carried out with the MEGA program. DNA-DNA hybridization was done using the membrane filter method described by Ezaki et al. with biotin-labeled chromosomal DNA from Pseudomonas sp. strain WO24, P. mexicana JCM11524 and P. japonensis JCM11525. Extent of hybridization was determined by ELISA using streptavidin-HRP and ABTS (E2,2′-azinobis[3-ethylbenzothiazolin-6-sulfonic acid]). [...] Homologs of DAP BII, PgDPP7, PgDPP11 and SpDPP11, were found by PSI-BLAST analysis in the database of non-redundant protein sequences at the National Center for Biotechnology Information. Subsequent analyses were performed by the MEGA program. In total, 661 candidates for family S46 peptidases were filtered from a MUSCLE multiple sequence alignment, based on the presence of conserved His and Ser residues that form part of the catalytic triad. A phylogenetic tree was constructed for a selected set of family S46 peptidases by maximum likelihood analysis using a WAG +F +G +I substitution model which was selected from fits for 48 different amino acid substitution models.Secondary structures of family S46 peptidases were predicted using Jpred (http://www.compbio.dundee.ac.uk/www-jpred/), while secondary structures for known Clan PA peptidases were deduced from tertiary structures deposited in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/home); i.e. for Chymotrypsin A from Bos taurus (1YPH.pdb) with catalytic triad H-75, D-120, S-213, Tsh peptidase from E. coli, (1WXR.pdb) with triad H-125, D-153, S-259, and lysyl peptidase from Achromobacter lyticus (1ARB.pdb) with catalytic triad H-262, D-318, S-399. […]

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