LOC56985 discussion

Evolutionary analysis of LOC56985 suggest that this protein is inherited through vertical transmission, with only one one exception seen in Trypanosoma. The eukaryotic Trypanosoma was grouped in with the bacteria and still retained a relatively high bootstrap value of 75%. Further analysis of the Trypanosoma protein sequence showed that it's protien is has a considerably larger sequenece than all others. This finding may lead one to hypothesise about a possible insertion of the protein within another gene in Trypanosoma through means of lateral transmission.

Clustal alignment also showed various amino acid residues that are conserved throughout all species, suggesting an important role for these residues in the structure or function of the protein. (thats my bit on evolution for now, might add to it later.)

this is just a part of the first draft, i'll be putting more up soon. just post what you got here, i'll get rid of the rendundat bits and combine the whole thing

Our results provide a structural, phylogenetic, and biochemical basis for the functional annotation of the human gene Loc. These findings are also extrapolable to the multitude of hypothetical metallophosphoesterase orthologs recently identified in mammalian and plant genomes. However, there is minimal experimental evidence concerning loc orthologs currently available to ascertain cellular roles and substrate specificity by bioinformatic analysis alone. Large scale gene expression analyses, which demonstrate highly controlled and tissue specific expression of loc orthologs, suggest potential significance to disease states and warrants future experimental analysis.

SCOP and Pfam analysis of the loc sequence identified a lone metallphosphoesterase domain and hence classified it as a member of the metallophosphoesterase superfamily. This is a relatively well characterized superfamily which counts paps, DNA double-strand break repair nucleases and protein serine/threonine phosphatases among its constituent families. The superfamily server failed to assign loc to any established SCOP family (no E-value <0.01 was obtained).

Comparison of 2nxf structure to all protein structures currently available via the PBD showed that it is most similar to paps. As mentioned above, both paps and loc orthlogs are metallophophoesterases and hence are likely exhibit significant similarities in topology, fold organization active-site organization. The biochemistry behind metal binding and phosphoesterase activity is identical among all members of the superfamily. Each family and subsequently each family member is unique in its metal ion and substrate specificities.

It was confirmed that loc is not a member of the pap family by homology search using blastp and sequence comparison of homologs using clustalw2. Whilst all PAPs and loc orthologs contain the GNH pattern which identify them as metallophosphoesterases, they show no other sequence similarities. In contrast, additional conserved patterns are present in all loc orthologs of vertebrates, plants and green algae. Likely prokaryotic precursors of loc orthologs were also identified in proteobacteria and xxxbacteria . Even though these prokaryote homologs do not exhibit all the conserved patterns seen among vertebrates, plants and green algae, they still exhibit greater homology to loc than any paps. Loc ortholog expression is limited to vertebrates, plants and green algae, whereas, paps are a widespread metallophosphoesterase family with well characterized members in all eukaryotic phyla and a larger variety of prokaryotes than loc orthologs. Loc orthologs are potential candidates for a novel metallophosphoesterase family.

Until very recently, there was no experimental evidence for the cellular role, and substrate and metal ion specificities of loc orthologs.

Whist Zn ions were used as ligands during crystallization of 2nxf for structural studies, the in vivo metal ions which ligand with 2nxf was unknown. It is likely that loc orthologs are specific for Mn (II), as shown in canoles et al., where the rat ortholog failed to activiate in the absence of Mn(II). The putative active centre of loc is within the cavity where PO4 lies bound to a pair of metal ions as shown in the crystal structure of 2nxf. Protein cavity prediction using CastP identified the PO4-bound cavity as the largest in loc. cavity residues which stabilize the metal ions and bind PO4 are also likely to be involved in the catalytic step. For example, Hisxxx (part of the catalytically relevant GNHE region in all metallophosphoesterases) interacts with the oxygen of the phosphoanhydride linkage which is duly hydrolysed.

Substrate specificity should be accounted for by loc residues which interact with the non-phosphate regions of the substrate. For instance, the crystal structure shows a smaller cavity on 2nxf which binds an ethanol molecule. Sequence alignments show that residues that directly bind EOH at this cavity are conserved among all loc orthologs. It must be noted that the PO4 complexed to 2nxf is not a complete representation of the enzyme-substrate complex. It is rather a potential representation of PO4 bound to the enzyme subsequent to the catalytic step.

Loc orthologs show no sequence or structural similarities to Nudix hydrolases.

The structural homology results obtained from the Dali server showed strong homology between the protein studied here and purple acid phosphatases. Purple acid phosphatases have been characterised as enzymes that catalyse phosphate ester hydrolysis under acidic conditions, are ~35kDa in size and contain two Fe ions at their catalytic centres (Wilcox, 1996). Similar acid phosphatases found in kidney bean, sweet potato and other plants contain Zn and Fe, two Fe or Mn at their cores.

Although the physiological role of purple acid phosphatases in the cell in not known, some of the proposed biological functions include iron transport, generation of reactive oxygen species and bone resorption (Oddie, et al., 2000); plant functions may include phosphate metabolism and generation of reactive oxygen species (Leung, Teixeira, Guddat, Mitić, & Schenk, 2007). Their localizations in the lysosomes of osteoclasts and macrophages support an intracellular monophosphate function in animals (Doi, Antanaitis, & Aisen, 1988). What is interesting from the results obtained here is that the ortholog from Zebrafish does not contain the characteristic iron core. The structure shows two Zinc ions at the catalytic site with the phosphate ion ligand. This may be the first case where a purple acid phosphatase does not contain this iron core.

Abstract | Introduction | Results | Discussion | Conclusion | Method | References

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