DAP discussion

Discussion

Aspartyl aminopeptidase (DAP) is defined as having a preference for N-terminal aspartyl and glutamyl residues but is distinct from glutamyl aminopeptidase. It is expressed in all tissues at a relatively high level representing >0.1% of soluble protein in cells, Wilk et al. (1998). It is also expressed at fairly equal levels throughout human tissue; this indicates a basic role in protein metabolism within cells.

sequence analysis of the protein taken from pseudomonas aeruginosa places i in the M18 family of the MH clan of cocatalytic metallopeptidases. It has been classified as aspartyl aminopeptidase and its crystal structure was resolved.

Very few aspartyl aminopeptidases have been characterised to date so little is known of the function of these enzymes. They are believed to be widely distributed in eukaryotes and prokaryotes. The function is to cleave amino acid residues at the N-terminus of unblocked peptides. To date yeast, human and the malarial parasite aspartyl aminopeptidase have been extracted and characterised. Genome sequencing has identified several other homologues however they are yet to be confirmed experimentally.

Many families of proteins within MH clan of metallopeptidases are characterised by the zinc binding motifs such as HEXXH however it is now known that no such motif exists for M18 family proteins, Yokoyama et al. (2005). Due to very little knowledge about that active sites of these proteins its catalytic centres are characterised based on sequence homology which has yielded a small selection of highly conserved His, Glu and Asp residues which are known to be involved in zinc binding. Two of three His residues that are considered to be fully conserved across M18 members are thought to be involved in the coordination of Zn2+ ions at the active site as mutation of these residues results in abolition of enzymatic function, Wilk et al. (2002).

To identify or predict the structure, function and evolutionary history of DAP many tools were utilised. P. aeruginosa DAP is composed of 12 identical subunits each with 428 residues. A DALI search was able to identify structural similarities with the current DAP chain-A and other aminopeptidase moleules, see fig. X.

To predict the function of P. aeruginosa DAP an InterPro scan identified the sequence as Aminopeptidase I zinc metalloprotease (M18). It listed 6 motifs of commonly conserved regions within the family. Based on the two papers by Wilk et al. (1998) and Wilk et al. (2002) Glu, Asp and His residues that were conserved and predicted for catalytic function were identified in the P. aeruginosa DAP. These residues mostly fell into these nominated motif regions indicating that the active sites were present there. A structural alignment between mouse and human with P. aeruginosa DAP showed that all the nominated residues were fully conserved between the species, making P. aeruginosa a good candidate to study DAP in a bacterium. A CluSTr analysis of the sequence was more accurate and figures X-X can be seen in the results. It predicted a probable M18 family aminopeptidase 2 and gave three potential zinc binding residues His82, 156 and 401 which correspond directly to the human residues predicted in the aforementioned paper.

Analysis of chain-A structure reveals that the residues of the predicted active sites cluster together (see fig. X). This indicates that the predictions are correct however analysis of the whole structure is needed to confirm this.

Viewing the current DAP in PyMoL reveals a dodecameric (12 subunits) tetrahedral shape, this has been identified previously in other members of the MH clan such as the FrvX homolog in P. horikoshoii, Russo & Baumann (2004). This protein features a catalytic zinc centre where substrates can access the active site through four channels in the centre of each triangular face of the dodecameric tetrahedral structure. This ensures that small or completely unfolded proteins can act as substrates.

One of the problems with determining the function of a crystallised protein is whether it dissociates in vivo to perform its biological function or whether the crystal structure is maintained and is necessary for activity. The paper by Wilk et al. (2002), provides evidence that DAP retains a three dimensional structure as it proposes that His352 is important for subunit-subunit interactions. Mutation of this residue dissociates the enzyme and catalytic function is lost implying that oligomeric DAP is catalytically active. This paper was however published before the crystal structure for DAP was resolved and it states that assignment of function to the histidines analysed awaits the solution of the crystal structure. Using PyMoL to analyse the named His352 residue or His313 in P. aeruginosa in the crystal structure of DAP shows that the residue is located between adjacent subunits (fig. X) indicating a structural role.

Similarly Histidines 349, 359 & 363 in the human homologue were experimentally shown to reduce kcat but not km so the paper states these also may contribute to stability. However structural analysis shows that this cluster is located in places around a single channel in each face of the tetrahedral structure. This indicates a role in guiding the substrate through the channel to the active site. So the reduction in kcat is probably due to the reduced likelihood of the substrate coming into contact with the active site.

To verify the above assertion the nominated active sites His94 and His440 were analysed in the crystal structure and it was found that they were distributed in the internal part of the structure (fig. X). This indicates a similar system to that of the FrvX homolog in P. horikoshoii mentioned above.

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