Computational protocol: Impaired coordination of nucleophile and increased hydrophobicity in the +1 subsite shift levansucrase activity towards transfructosylation

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Protocol publication

[…] Individual datasets were created for GH family 32 and 68 by extracting all sequences (7544 and 1155, respectively) from UniProt. Identical sequences were clustered and the datasets were further minimized by reducing the redundancy to 95% using BLASTClust (https://toolkit.tuebingen.mpg.de/blastclust) () to allow Clustal Omega () (limit: 4000 sequences) and MUSCLE () (limit: 500 sequences) alignments ().Family GH32. Sequences with less than 400 and more than 700 amino acids and sequences containing unspecified amino acids (“X”) or that were not starting with methionine were removed to further minimize the dataset using BioEdit to yield a dataset with a total of 3651 sequences. A Multiple Sequence Alignment was created with Clustal Omega. The corresponding amino acid positions were identified using the structure similarity tool provided by the PDB-database (http://www.rcsb.org). Amino acid distributions for the positions of interest were analyzed with the positional frequency tool (BioEdit Version 7.0) ().Family GH68. Sequences with less than 350 amino acids, containing unspecified amino acids (“X”) or not starting with methionine were removed to further minimize the databases using BioEdit. The sequences for Bm-Ls (D5DC07) and Streptococcus oralis Ls (P05655) were added manually to the dataset since they were deleted by BLASTClust. From this dataset (367 sequences), a local BLAST database file was created using BioEdit and Local BLAST 2.0 () (internal version in BioEdit 7.0) was performed using Bm-LS sequence (D5DC07) as query. A Multiple Sequence Alignment was created with the MUSCLE webserver and the resulting MSA was sorted by the scoring list from Local BLAST. Amino acid distributions for the positions of interest were analyzed via the positional frequency tool (BioEdit 7.0) (Supplementary data, Table SI). […]

Pipeline specifications

Software tools BLASTclust, Clustal Omega, MUSCLE, BioEdit
Applications Protein sequence analysis, Nucleotide sequence alignment
Organisms Bacillus megaterium
Chemicals Hydrogen, Sucrose, Tyrosine