Arylformamidase Results
most similar sequence - catalytic triad, structure with highlighted
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).
most similar structure - catalytic triad, structure with highlighted
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.
Sequence & Homology
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).
Figure 1.
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).
Figure 2.
Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.
Structure of Arylformamidase
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.
Figure: Arylformamidase (All Chains)
The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). These ions aren't biologically significant and could only be an artefact. When crystallizing proteins they often form complexes (dimer, tetramers etc) but that doesn't mean that the functional structure is the same. They could be functional monomers. The chains in the protein of interest exist as individual functional units because in the PDB file it assumes the functional biological molecule as a monomer.
Image from PDB ProteinWorkshop 1.5
Figure:Chain A of arylformamidase
The red molecule in the middle is an unknown ligand containing a ring composed of 9 oxygen molecules. The green sphere is a chloride ion.
Image from PDB ProteinWorkshop 1.5
The protein backbone is coloured by conformation type:
Turn - blue
Coil- pink
Helix- green
strand- purple
Interaction of human arylformamidase (AFMID) with other proteins
The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:
1- Neighborhood in the genome
2- Gene fusions
3- Cooccurence across genomes
4- Co-Expression
5- Experimental/Biochemical data
Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins
TM1040_2226 Tryptophan 2,3-dioxygenase (279 aa)
TM1040_2225 Kynureninase (396 aa)
TM1040_2493 Succinic semialdehyde dehydrogenase (490 aa)
TM1040_1862 Hypothetical protein (212 aa)
TM1040_2491 Creatinase (402 aa)
TM1040_2736 Transketolase, putative (794 aa)
There is no significant evidence for these interactions (score= ~0.5)
DALI OUTPUT
The DALI tool produces proteins that are structurally similar to the protein of interest.
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.
Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)
PDB link title
Note: Chain B not shown
From PDB ProteinWorkshop 1.5
Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)
File:Carboxylesterase (archaeon).txt
PDB link title
Note: Chains B, C & D not shown
From PDB ProteinWorkshop 1.5
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.
Secondary structure analysis
PDBSum output for arylformamidase
PDBSUM [1]
Figure: Archeon Carboxylesterase secondary structure
The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.
Images from PDBsum
The conservation of the ser/his/asp catalytic triad
Yellow indicates conservation
Blue indicates semi-conservation
Figure: The catalytic triad
The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. Note: The actual residue numbers are n+1
Image generated using Pymol
Figure: The conserved residues of arylformamidase
The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.
Image generated using Pymol
Figure: The catalytic triad
The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)
From PDB ProteinWorkshop 1.5
Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)
The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.
