One gene our team will be using as a node in our Hamiltonian Path problem is Kanamycin resistance translated in the form of Kanamycin nucleotidyltransferase (KNTase). The antibiotic Kanamycin, once in the cytosol of E.Coli, inhibits protein synthesis by interacting with the “decoding” region in the small ribosomal subunit RNA.(Sambrook and Russel, 2001) The KNTase enzyme, as a member of the aminoglycoside phosphotransferase (APH) enzyme family, blocks Kanamycin’s ability to inhibit protein synthesis by transferring a nucleoside monophosphate (adenyl) group from Mg2+-ATP to the 4’ hydroxyl group of Kanamycin, inhibiting its ability to bind to the srRNA. [http://www.ingentaconnect.com/content/els/00452068/1999/00000027/00000005/art91144 1]

Our goal was to insert a hix site (a polar molecule) in an area of KNTase protein that would not interfere with its ability to inhibit Kanamycin. We looked at mutational analysis of KNTase and other aminoglycoside phosphotransferase enzymes to determine which aspects of KNTase’s structure were integral to its function and therefore not an ideal site for hix site insertion. KNTase is a dimer consisting of 253 amino acids in the molecule [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 3]. In looking at conserved structures in the APH family we took into consideration that:

-Substitution of AA 190 caused 650-fold decrease in enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]

-AA 190 is involved in catalysis [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]

-AA 195 and 208 are involved in Mg2+ binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htmv 5]

-Mutant Enzymes 190, 205, 210 all showed changes in mg+2 binding from the WT [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]

-Substitution of AA 210 (conserved) reduced enzyme activity [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]

-AA 166 serves to catalyze reactions involving ATP [http://www.bioscience.org/1999/v4/d/perlin/fulltext.htm 2]

-AA 44 is involved in ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]

-AA 60 is involved in orientation of AA 44 and ATP binding [http://www.bioscience.org/1999/v4/d/wright/fulltext.htm 5]

-We did not consider any Amino Acids near the N or C terminus

-We did not consider any residues near ß-sheets or ∂-helices close to the active site because hydrogen bonding plays an active role in substrate stabilization and the polarity of our hix site could disrupt the secondary structure and therefore the hydrogen bonding ability of KNTase)

We decided to insert our hix sites at the 125 AA of each monomer due to their distance from each other, active site secondary structure, N or C terminus, and lack of any previous mutational analysis proving its function as integral.

We use genes to represent the nodes on our Hamiltonian path. One of the essential features of these genes is that they can tolerate the insertion of a Hix site. It has been previously demonstrated that GFP fluoresces despite a Hix insertion. Another glowing protein, [http://partsregistry.org/Part:BBa_E1010 RFP] (from [http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=86600 Discosoma sp.]), is a candidate for use in our path. Although its DNA sequence is markedly different from GFP's, it has some amino acid similarity and a remarkable structural similarity. Both proteins have a Beta-barrel structure which surrounds an internal chromophore.

Inserting 13 amino acids into a protein can potentially disrupt its ability to function. It is thus essential to find an insertion point that does not interfere with the protein's function. Fortunately, the similarity between GFP and RFP allows us to make a highly educated guess for where to insert. RFP's amino acid position 154 is homologous to GFP's amino acid position 157, which is where GFP was split. This is therefore our best guess for where to insert the Hix site.

The specific Chloramphenicol Acetyltransferase gene we are using comes from the plasmid PSV2CAT whose original source is an E. coli transposable element Tn9 (Sambrook, 2001) Its PDB ID# is [http://www.pdb.org/pdb/explore/explore.do?structureId=1PD5 1PD5].

I have chosen to insert my hixC site between amino acid 52 and 53. I chose this point because it is away from the active site of the protein, the point that contains the catalytic binding sites and allow the recognition and binding of the substrate. It is important for the insertion point to be away from the active site because we do not want the overall function and structure of the protein to be destroyed in the process of splitting. We want to split at a point where the two halves of the protein cannot work as single units, but once a hixC site has been inserted, and the two halves are brought back together, the protein displays its original function.

In researching Cre Recombinase, we found that the gene had already been split by another lab. [http://www3.interscience.wiley.com/cgi-bin/abstract/104558885/ABSTRACT] In the study done by Casanova et al, two independent but overlapping sections of the Cre Recombinase gene were placed in separate locations along an E. Coli chromosome. When translated, the two overlapping halves of the Cre Recombinase protein bound together and formed a functional Cre Recombinase protein.

In order for Casanova et al's split protein to be functional, the overlapping section of the bound protein at the split site presumably could not significantly hinder the protein’s ability to bind to loxP sites or recombine DNA segments. With that in mind, we investigated the same region split by Casanova et al. as the prime candidate for the insertion of a hixC site.

The site that was eventually chosen reflects both the protein structure shown to the right and the previous research done in the Casanova lab. We believe that amino acids 190-191 along the Cre Recombinase protein are unlikely to play a significant role in the functioning of the protein, thus we decided on this location for the insertion of our hixC site.

Figure 1 on the right depicts a monomer of Cre Recombinase bound to a DNA strand that it is about the cut. Our split site is highlighted in yellow and can be seen far away from the active site of the molecule.

Figure 2 on the right depicts two dimers of Cre Recombinase coming come together to cut DNA at two loxP sites. The site of our hixC insertion is highlighted in yellow on each molecule and can be seen far away from the active site.

Figure 1

Figure 2