Computational protocol: Discovery of a Eukaryotic Pyrroloquinoline Quinone Dependent Oxidoreductase Belonging to a New Auxiliary Activity Family in the Database of Carbohydrate Active Enzymes

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Protocol publication

[…] The C. cinerea genome database (http://www.broadinstitute.org/annotation/genome/coprinus_cinereus/MultiHome.html) was searched with the amino acid sequence of CDH from P. chrysosporium (accession no. AAB92262) using the TBLASTN algorithm , . The search was carried out with standard settings and the BLOSUM 62 matrix. The amino acid sequences of candidate genes were scanned for the presence of signal peptides using SignalP version 4.1 software at the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/) and searched against the protein database at the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The homology structure model was constructed using Protein Homology/analogy Recognition Engine (Phyre) version 2.0 (http://www.sbg.bio.ic.ac.uk/phyre2/) . The CcSDH model was used for preparing a figure in which PQQ of sGDH from Acinetobacter calcoaceticus and heme in the cytochrome domain of P. chrysosporium CDH (PDBID 1CRU and 1D7C, respectively) were superimposed to the model. The structure based alignment was created during the process of homology modeling using the Phyre2 server, and the corresponding figure was prepared using ESPript ver. 2.3 . [...] The National Center for Biotechnology Information protein database was searched for amino acid sequences corresponding to the catalytic domain of the recombinant protein using the PSI-BLAST algorithm. All searches were performed with standard settings and the BLOSUM 62 matrix. The sequences obtained and the sequences of known quinoproteins (PQQ-dependent methanol dehydrogenases, ethanol dehydrogenases, soluble glucose dehydrogenases, quinohemoprotein alcohol dehydrogenases, polyvinyl alcohol dehydrogenase, membrane-bound glucose dehydrogenase, membrane-bound alcohol dehydrogenase, and aldose sugar dehydrogenases) were subjected to alignment analysis using MAFFT (ver. 6.85) , . Non-conserved regions were trimmed using SeaView (ver. 4.4.2) software. The phylogenetic tree was generated from the trimmed sequences using ClustalX (ver. 2.1) with the neighbor-joining method (Bootstrap value, 1000). […]

Pipeline specifications

Software tools TBLASTN, SignalP, Phyre, ESPript, BLASTP, MAFFT, SeaView, Clustal W
Organisms Homo sapiens, Coprinopsis cinerea
Chemicals PQQ Cofactor