MacDNAsis of Hexokinase

Results of MacDNAsis analysis of hexokinase

MacDNAsis is a computer program which can analyze DNA, RNA or amino acid sequences to give a variety of information. The following information was collected based on the yeast mRNA sequence for hexokinase, which was found from Genbank.

determination of the largest open reading frame
Sequence information: molecular weight
Structural information: Kyte and Doolittle plot, Hopp and Woods plot, secondary structure
Comparison among species: multiple sequence alignment and phylogenetic tree

1. Determination of the largest ORF

To start the analysis of hexokinase, the mRNA sequence of this protein in yeast was used to determine the ORF (open reading frame) that encoded the protein.

Figure 1: ORF for yeast hexokinase mRNA. Red triangles indicate start codons, green lines indicate stop codons. The black box represents the largest ORF for the mRNA sequence. Each row represents a different reading frame and the resulting start and stop codons. The ORF covers nucleotides 718 to 2178.

2. Sequence Information

The ORF of yeast hexokinase mRNA was translated by MacDNAsis to give the amino acid sequence. The program calculated the molecular weight to be 53,949.57 kD. This sequence was used to predict secondary structure of hexokinase and to prepare hydropathy and antigenicity plots.

3. Structural Information

The amino acid sequence was used to predict structural information about hexokinase. Three types of analysis were performed for hexokinase: a Kyte and Doolittle hydropathy plot, a Hopp and Woods antigenicity plot, and a Chou, Fasman and Rose secondary structure prediction.

Kyte and Doolittle hydropathy plots predict whether the protein is a integral membrane protein. Negative numbers represent hydrophilic regions; positive numbers represent hydrophobic regions. A value greater than +1.8 suggests where transmembrane regions may lie.

Figure 2: Kyte and Doolittle hydropathy plot for hexokinase. This plot suggests there is one segment that crosses a membrane near amino acid 400. Most other regions of the protein appear to be hydrophilic.

The Hopp and Woods antigenicity plot predicts areas that may make good epitope sites. These sites are characterized by greater hydrophilicity. Since hydrophilic regions are usually found on the surface of the protein, the amino acids are better exposed to potential antibodies. Hydrophilic regions are denoted by positive numbers; hydrophobic by negative numbers.

Figure 3: Hopp and Woods antigenicity plot for hexokinase. No major hydrophilic regions are evident; however, the overall structure appears to be more hydrophilic then hydrophobic. This prediction agrees with that of the Kyte and Doolittle plot.

The Chou, Fasman and Rose function in MacDNAsis predicts the secondary structure of proteins in a simplified linear form. The analysis predicts a-helix, b-pleated sheets, turns and coils.

Figure 4: Predicted secondary structure of yeast hexokinase. The tertiary structure can be viewed with RasMol.

4. Comparison among species

MacDNAsis was used to compare the hexokinase amino acid sequence translated from yeast mRNA to the amino acid sequence found for four other species. Genbank searches for Schistosoma mansoni, Arabidopsis thaliana, mouse and human provided amino acid sequences for hexokinase in each species. (To view the results of that search, click on the corresponding species.)

Multiple sequence alignment was performed to determine the genetic conservation among the five species. A sample of the alignment is shown below.

Figure 5: Segment of amino acid sequence of hexokinase for each of the five species analyzed. Blackened letters indicate amino acids that match the yeast sequence.

A phylogenic tree calculating the degree of homology between species was generated after the sequence allignment.

Figure 6: Phylogenetic tree for the five species in this analysis. The degree of amino acid conservation between species is indicated by percentage values. Strong conservation is seen between the human and mouse sequences (89.6%).

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