Stevens: Malic Enzyme

Zea mays	Arabidopsis thaliana

The enzyme that I selected to study for the protein project is NADP+-malic enzyme. One of the main uses of this enzyme in plants is in C4 and CAM carbon assimilation pathways. These two pathways serve to help eliminate the oxygenase activity of RuBisCO, and thus prevent photorespiration, by concentrating carbon dioxide around the active site of RuBisCO. NADP+-malic enzyme is one of the three decarboxylating enzymes used in the C4 and CAM pathways. Malate is the four carbon sugar that is decarboxylated by NADP+-malic enzyme to concentrate CO2 around RuBisCO.
In my research, I am investigating the means by which the macroalga Fucus vesiculosis is able to continue photosynthesis for a finite period of time in a CO2 free/ high O2 environment. Prior research has shown that there is a stored organic pool of carbon that the Fucus spp. utilizes for photosynthesis in a CO2 free environment. It has been hypothesized that this stored organic pool consists of malate. My research will involve trying to determine the exact components of the organic pool. NADP+-malic enzyme could be the protein that decarboxylates malate, assuming malate is a main constituent in the stored organic pool.
In this project, I compared NADP+-malic enzymes from Zea mays, a C4 plant that is a monocot, and Arabidopsis thaliana, a well-studied dicot. The percent identity in nucleotide alignment was 65.8 %. Peptide similarity was 78.6% and identity was 71.5%. The protein structures were analyzed using 3D-PSSM and models were returned showing the tertiary structure with only the alpha carbon backbone (i.e.-no side chains). The protein structures were fairly similar. Main differences were seen in similar structures with completely different amino acids, and in regions where one protein lacked sequence where the other had sequence.

Both the proteins do not start directly at the beginning of the peptide sequence. Instead, arabidopsis starts at peptide 93 and corn at peptide 120. Additionally, the arabidopsis sequence skips from residue 100 to 112, thus removing a gap in the alignment of the peptide sequences. Though these start structures look very similar, they are quite different in their amino acid sequence.
CORN: F-Y-I-S-G-Y-T-L-L-R-P
ARABIDOPSIS: E-D-M-P-I-T-P-W-R-D-P
Some of the amino acids in these sequences are similar, whereas most are completely unrelated. This is another region where again, the structures look the same, but the amino acids are different. This region occurs in corn between 140-143, and in arabidopsis between 123-126.In this case, the amino acids that are different are similar. (note- numbers run from right to left)
CORN: M-Q-K-Q
ARABIDOPSIS: S-H-R-Q

Again, the region looks very similar, but there is a difference in the amino acids. This section occurs in corn between 235-240, and in arabidopsis between 218-231, with a skip from 220-227. This skip of amino acids in the arabidopsis sequence coincides with a lack of amino acids in the alignment of the two sequences. There is some similarity between the amino acids of the two proteins.
CORN: S-L-R-D-K-G
ARABIDOPSIS: S-L-N-N-R-G

This region of difference is the beginning of a major divergence between the two structures. The arabidopsis sequence occurs between 245-252 and the corn is between 254-261. In the alignment of peptide sequences, there is a gap in the arabidopsis sequence where corn has residues 255-273. This is a point of divergence, as will be seen, because the corn protein includes those amino acids, whereas the arabidopsis model does not have this region.
CORN: I-Q-V-I-C-V-T-D
ARABIDOPSIS: I-Q-G-M-G-I-P-V

The red section is the previously described divergence section, continuing on beyond the previous region. The main difference in the red section is the addition of an alpha helix structure that is not seen in the arabidopsis model. This additional structure is due to the sequence in corn that is lacking in arabidopsis. Once into the green section, you can see how the structures regain similarity.This display continues the above display, and shows how the models truly have regained similarity (yellow regions) after the divergence and addition of an alpha helix in corn. Overall, there are no major differences between the two models for the remainder of the structures.