Arylformamidase Methods
Literature search
A literature search was performed using the putative annotation ‘arylformamidase’. A paper by Pabarcus et al. 2007 was returned which, ironically, described the arylformamidase from Mus Musculus.
Conservation of Catalytic Triad
An alignment of 2pbl and the protein of interest was performed using ClustalX. Default parameters were used. Residues of the catalytic triad were identified from the paper describing it and located in the sequence. Conservation of the residue and the surrounding sequence was observed. Note: in analysing conservation of the 2c7b catalytic triad with 2pbl, the clustalW alignment was found to differ from the alignment provided as part of the DALI results.
Sequence & Homology
Using the query sequence Arylformamidase,a BLASTP search was performed on the bacterial protein sequence using a non-redundant database. The top scoring matches to an E-value of 3e-054, 35 sequences in total, were selected. Eukaryotic homologous sequences sequences were found using NCBI HomoloGene. These were appended to the list and a multple sequece alignment was performed using CLUSTAL X.
The data output from the multiple sequence alignment was bootstrapped 1000 times and a phylogenetic tree was created using the neighbour-joining algorythm. The program FigTree was used to create the visual representation of this tree.
A similar BLASTP search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.
Top scoring sequences from the results of the BLASTP search using the human homologue were appended to the original top scoring sequences of the results BLASTP search on the bacterial query sequence.
As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorithm.
Structure
A number of bioinformatics databases were used to obtain the structure of arylformamidase. These include RCSB Protein Data Bank (PDB ID: 2PBL)[1] for the quaternary protein structure and PDBsum [2] for the secondary structure.The predicted interaction of arylformamidase with other proteins was determined using the STRING database [3]. The DALI database was used for the structural comparison of arylformamidase with other proteins [4]. Pymol was used to make structure of arylformamidase showing the conserved catalytic triad.
