ACADM gene and it's disease

My pet gene for this semester is ACADM . It is a located at 1:76,190,042-76,229,354 (39,313 base pairs(bp)), containing 12 exons (1263 bp), which translates to a 421 amino acid (45 kilodaltons) protein. Because only 1263 bp are exonic and adding 6 bp per exon ( 3 for each side ) for splicing bases totaling 1335 (=1263+(6*12)) means that only a sparse 0.033% of the bases inside of this genomic region actually 'do something' to make the protein.

The protein itself encodes a dehydrogenase enzyme that degrades medium-chain fatty acids. Mutations resulting in a deficiency of the enzyme cause the, cleverly name, disorder Medium-chain acyl-coenzyme A dehydrogenase deficiency. I could find no reports of over production of the enzyme interestingly. The deficiency results in an "intolerance to prolonged fasting, recurrent episodes of hypoglycemic coma with medium-chain dicarboxylic aciduria, impaired ketogenesis, and low plasma and tissue carnitine levels. The disorder may be severe, and even fatal, in young patients" (Matsubara et al., 1986). For the assignment we will be modeling the variation for allele .0001 MCAD DEFICIENCY LYS304GLU created by the genomic variation dbSNP:rs77931234. This mutation may also be known as LYS329GLU (K329E) because the protein sequence itself is a precursor protein, in fact it will be the K at 329 we will need to change to an E in the sequence we use.

We will start by modeling the secondary structure of the reference protein sequence NP_000007.1, then the amino acid change LYS304GLU will be inserted where appropriate and again model the secondary change and compare the differences. The rs77931234 mutation occurs towards the beginning of of exon11 as seen in these UCSC screen caps (with rs77931234 highlighted in black). 

The protein sequence was loaded into DNASTAR's Editseq applications and Leucine(K) at position 304 (highlighted in black below) was changed to a Glutamic Acid(E). 

Here is a quick screen shot of the sequence alignment following the sequence change.

It isn't particularly surprising that this single amino acid change has a significant impact on the protein function;  Lysine is a strongly basic amino acid and Glutamic acid, the substitution, is a strongly basic amino acid.  The first step in comparing the two sequence is to load them into SeqBuilder and generate some statistics. To do this, the protein sequence was loaded into SeqBuilder, the entire protein sequence was selected, then by opening the "sequence" menu and clicking on "Statistics", we can determine the Isolectric Point, and the Charge of the protein at a PH of 7.0. For the reference sequence the Isolectric Point is 8.369, and the Charge is 5.546, this changes for the protein following the substitution of the Leucine with the Glutamic Acid to an Isolectric Point of 8.055, and the Charge at pH 7.0 is 3.550. Using these simple stats we can see that the mutation would have an impact on the enzyme at a basic bio chemical level. Unfortunately these high level statistics were the only metric I found that differentiated between the sequences.  Below are the sequences as the appear when loaded into the Protean tool in DNASTAT.  The black bar highlights the location of the mutation in each view.   The first is the reference sequence, the second is the mutated sequence.

NORMAL

MUTATED(LYS329GLU)

Using the algorithms available in the Protean tool sweet, no detectable impact on proteins structure was observed.  In both a high probability of the region of the mutation being part of an Alpha structure in the reference was no impacted by the presents of the mutation.  The Kyte-Doolittle Hydrophobicity plot (and probability) was unchanged by the mutation as well.  Because of the change in the charge of the overall protein, a further inspection of the hydorphobicity using a the Kyte-Doolittle algorithms and plots was carried out with the following plots observed, again with region of change highlighted in black, reference on top and variation on the bottom.

Again no appreciable change was observed.  The Chou-Fasman algorithm was used to inspect the region as well with the results send here:

While subtle probability shifts appear at adjacent locations no major structural change can be detection using these algorithms.   

While clinical evidence has repeatedly observed this mutation in the presence of low enzymatic activity and pathologic phenotype, none of the tools or algorithms were able to detect a major change in the protein structure caused by this variant. 

There are many other possibilities as to how this mutation could impact the function of this protein. As mentioned previously this protein sequence is actually a precursor protein and requires further editing and manipulation, this amino acid change could impact this reaction by blocking the catalytic site meaning the protein is never able to take on its fully functional form.  It is also possible that the way the protein binds to the fat molecule itself may be altered just enough to prevent the catalicataly activity of the enzyme.

As I learn to use more of the DNAstar tools I will continue to evaluate this mutation and attempt to untangle is impact.