DAP Evolutionary Analysis
Blastp results of the query sequence gi|119390187|pdb|2IJZ|A (Chain A, Crystal Structure Of Aminopeptidase) against non-redundant (-nr) database was selectively chosen for multiple sequence alignment using ClustalX 1.83.
Figure 2.1 Clustalx MSA part 1
Figure 2.2 Clustalx MSA part 2
Figure 2.3 Clustalx MSA part 3. Multiple sequence alignment of Aspartyl Aminopeptidase Chain A. Highlighted in blue box is the query sequence and red box highlights conserved and semi-conserved histidine residue
“Sequence of aspartyl aminopeptidase is highly conserved. For example, the human enzyme has 51% identity to an “aspartyl-aminopeptidase-like protein” in Arabidopsis Thaliana (Accession Number BAA97497)” –Wilk .et, al. 2002. This is true to our protein sequence which originated from Pseudomonas Aeruginosa.
Aspartyl aminopeptidase belongs to the M18 family of clan MH cocatalytic metallopeptidases [reference no 14 wilk 2002 paper]. The M18 family is widely represented in both eukaryotes and prokaryotes. It is not known how many of the other members of the M18 family are also aspartyl aminopeptidases since most of the entries in the databases derive from genome sequencing and the enzymatic activities of the encoded proteins have not been determined. Many M18 family members are highly homologous to aspartyl aminopeptidase, and it is likely that they are also aspartyl aminopeptidases (Wilk .et, al. 2002).
Sequence of the query aminopeptidase protein is highly conserved throughout multiple sequence alignment from the many other organisms, suggesting a fundamental role for this enzyme in cellular functions.
Figure 2.4 Conserved histidine residues from a Homo Sapien DAP query. Source: S. Wilk., et, al 2002
Figure 2.5 Manually curated conserved and important semi-conserved amino acid relative to gi:119390187, pdb:2IJZ_A sequences position sourced from ClustalX. To be used for structural analysis with Pymol
Among highly homologous eukaryotic sequences, 8 histidine residues were found to be conserved or semi-conserved throughout, namely histidine position: 33, 94, 170, 349, 352, 359, 363 and 440 (relative to Homo Sapiens DAP sequence). Through experimental means, these histidine residues were sequentially mutated to phenylalanine to explore functions of conserved histidines in DAP. These positions matched and existed in our query sequence 2IJZ_A , these histidines sequences along with other conserved reqion from ClustalX results were noted and used for structural analysis with Pymol.
Figure 2.6 Bootstrapped radial view of phylogenetic tree. Classified the life forms according to two-empire system, highlighted in red circle is prokaryota/procarya and in green is eukaryota/eucarya.
Figure 2.7Branch bootstrap values in rectangular cladogram view. Branches with strap value less than 75% were indicated with a red dot
In figure 2.6 which is the bootstrapped radial view of the phylogenetic tree created by Clustal, it could display a clear view of the compared protein sequence’s origins into two superkingdoms namely the Eucarya and Procarya. In the phylogenetic trees shown Procarya superkingdom could be further classified into Bacteria and Archaea domains, and in turn, the Eucarya could be sub-divided into 4 kingdoms which are: Protista, Fungi, Animalia and Plantae. Placement of the kingdoms are better displayed on figure 2.7, in which each species/organism is correctly displayed with its neighbouring counterparts. Every species are in their rightful groups, which means that the DAP encoding genes are most likely to be vertically transferred, and not laterally. With the knowledge of important functions of DAP, such as to remove glutamyl or aspartyl residues from N-terminal acidic amino acid-containing peptides, it could be concluded that the protein itself widely distributed and essential for functional purposes.
