Phytanoyl-CoA discussion

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

Evolution Discussion

Whilst the phylogenetic tree seems to groups similar organisms together well there were some unexpected placements. Fungi were related further from plants than what might be expected. This supports the fact that fungi are more closely related to animals than plants as theory suggests, however it also shows that the PhyD must be required far more by these types of organisms than in plants. Due to the function of this protein being to catalyze alpha oxidation of fatty acids, it can be inferred that organisms feeding on tissues containing such fatty acid would require phytanoyl-CoA dioxygenase more than other organisms such as plants. An example of such a fatty acid is chlorophyll, meaning that all organisms trying to digest plant matter would require a functional gene to deal with its breakdown. This is then why high conservation has been maintained in the animal and fungi domains but not a greatly in plant and bacterial organisms.

The two protozoan have been grouped differently in the original and boot-strapped trees an both have boot-strap values below 50% indicating that the grouping is not a highly supported one. In particular the grouping of the Monosiga brevicollis has a boot-strap value of 29% which is too low to place any confidence in its position. The grouping of Tetrahymena has a higher value which indicates that there may be the possibility that lateral gene transfer has occurred. Tetrahymena is a phagotrophic protozoan which inhabits mainly fresh water and can exist in both commensualistic and pathogenic modes. Lateral gene transfer is rare among eukaryotes however the its frequency among phagotrophic protozoans is comparable to that of prokaryotes. Thus due to the environmental niche that Tetrahymena exists in, it is quite possible that leteral gene transfer occurred between it and another water dwelling animal.

Structure Discussion

The structure was found using vapor diffusion (hanging drop) crystallography. It was found through secondary structure comparisons that the most similar protein was Phytanoyl-CoA 2 Hydroxylase. The structure was shown to contain a large cleft on one side, this was shown to be the location of the binding sites for Fe(II) and the oxogluturate ligands. This agreed well with the observations made.

The structure itself contained several beta sheet regions, the largest of these wrapping around the core.

Isoform a itself was shown to be most similar to isoform b. Isoform c is the most different of the 3 due to a change in reading frame.

The information detailed in the structure section was shown to correlate well with function and evolutionary characteristics.

Function Discussion

After determining that the unknown protein was a member of the phytanoyl-CoA dioxygenase family of enzymes by InterPro analysis, it was compared to the the only other determined structure in the family, PhyH (PDB code 2a1x). The iron binding site of PhyH is composed of residues PRO173, HIS175, GLN176 and ASP177 (Searls et al., 2005) show structural similarity to the PRO155, HIS156, GLN157 and ASP158 of PhyD. HIS220 is also involved in iron binding in the hyroxylase but no histadine residue exist in this position in PhyD. It is hypothesised that the histadine binding is replaced by a serine at position 160, which is spacially close to the histadine in the pymol alignment. Similarly, the 2-oxogluterate binding site of PhyH (residues TRP193, ILE159 and ARG275) could be overlayed with residues TRP174, ILE143 and ARG257 of PhyD. Upon alignment of the whole proteins, the spacial positions of of the 2-oxogluterate and iron binding sites were highly conserved in the largest cleft of both proteins (figure 3.5). It can be inferred that PhyD is a catalyst for the oxidation of phytanoyl-CoA due to the structural similarities with PhyH.

PhyH is a peroxisomal enzyme synthesised as a pro-protein containing a type-2 N-terminal peroxisomal targeting sequence (PTS). The PTS-2 is recognised by the PEX-7 receptor, localised in the peroxisomal membrane, and transported into the peroxisome. Once in the matrix the PTS-2 signal is cleaved between THR30 and SER31 to produce the mature enzyme (MaDonough et al., 2003).

A sequence scan of PhyD failed to find a N-terminal PTS-2, which is charecterised by the sequence (R/K)(L/V/I)X,,5,,(H/Q)(L/A) in the first 20 amino acids (Pertriv et al., 2004). Nor was a PTS-1 sequence found at the carboxyl end. The PTS-1 sequence is usually charecterised by the tripeptide SKL at the extreme C-terminal, however through mutations in the PTS-1 sequence of luciferase, Gould et al. (1989) found that the protein was still targeted to the peroxisome with the sequence (S/A/C)(K/H/R)L. All combinations of PTS-1/PTS-2 sequence were searched for in the sequence of PhyD but none were found. This result means that it is unlikely that PhyD interacts with the peroxisomal receptor proteins PEX-5 and PEX-7 to gain entry to the peroxisome. PhyHD may still take part in alpha-oxidation as many peroxisomal proteins have been found to enter the peroxisome in an uncharecterised manner (ref....)

No interaction partners for human PhyD, or the homologous protein from other species, were found via STRING analysis. PhyH interacts with several proteins including PEX7 and coagulation factor VIII, but no inferences about PhyD can be drawn from this due to the low sequence homology.

The expression data obtained from Genomics institute of novaris research foundation (http://symatlas.gnf.org) indicated a difference in tissue transcription between PhyH and PhyD (figures 3.6 & 3.7). PhyH is expressed highly in the liver, which is expected as long-chain fatty acids are delivered from the gastrointestinal tract, via the portal vein, to the liver for processing. A defect in PhyH produces phytanic acidaemia, an increase in blood phytanic acid due to inefficient processing in the liver. This is a symptom of Refsum's disease, a recessive genetic disorder caused by mutations in PhyH or the transproter PEX7. Interestingly, the expression of PhyD is highest in tissues that are symptomatic in Refsum's. The presence of increase PhyD expression may indicate that these tissues are particularly sensitive to phytanoyl-CoA/phytanic acid. The phytanic acidaemia induced by a dysfunctional PhyH/PEX7 gene could overload the alpha-oxidation pathway in these cells culminating in cell death. High expression of PhyD in the dorsal root ganglia and spinal cord can be directly linked to the sensory neuropathy associated with Refsum's whilst high expression in the olfactory bulb could account for the anosmia.

Genetically, Refsum's is recognised as a heterogenous disease with up to 55% of cases not being linked to the PhyH gene (Weirzbicki et al., 2000). This indicates that the disease may be linked to several members of the phytanoyl-CoA dioxygenase family of their transport proteins.

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


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