Team:Nevada/Modeling

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University of Nevada, Reno

We developed a computer model for measuring enzyme kinetics of our pathway (Figure 1). The model was developed using Mathematica based upon steady-state Michaelis–Menten kinetics (Figure 2). It revealed that the kinetic parameters for the wild type isoform of 4-coumarate:CoA ligase were creating a bottleneck and significantly slowing down the overall reaction (Figure 3). Our group postulated and tested two potential solutions to get around this problem: (1) to use a mutant enzyme (Figure 4) with more favorable kinetic parameters or (2) To increase the concentration of the wild type isoform of 4-coumarate:CoA ligase (Figure 5). The mutant isoform was selected for based on a 2000 paper by Stuible, et al. at the Max Planck Institute who altered three polar amino acids into hydrophobic groups and discovered the mutant's affinity for the nonpolar sites of cinnamic acid increased dramatically (3). For all of the kinetics characterized assume all of the enzymes are in equal concentrations, except for the model involving 10x the concentration of the wild type isoform of 4-coumarate:CoA ligase (Figure 5). We chose to investigate both of these methods to insure no ill-conceived reaction kinetics were occurring.

While we have a hypothetical model for the reverse reaction, it is purely speculative as no real world data exists characterizing the reverse reaction rates of the aforementioned enzymes. This also means our model does not account for reverse reaction kinetics due to the lack of available data. We also used kinetic values from a highly conserved variant of Cinnamoyl-CoA reductase called Feruloyl-CoA reductase. Data for the latter was obtained from the Arabidopsis thaliana isoform and is structurally similar enough to act as a proper substitute for Cinnamoyl-CoA reductase (Table 1) Our model has proven to yield vital information regarding the proper kinetic parameters our team needs to examine in order to establish an efficient, and effective pathway. .

Figure 1 - Cinnamaldehyde Synthesis Pathway

Figure 2 - Steady State Model of Cinnamaldehyde Production from an L-Phenylalanine Precursor using the wild type isoform of 4-coumarate:CoA ligase

Figure 3 - Steady State Model of Cinnamaldehyde Production from an L-Phenylalanine Precursor using the mutant isoform of 4-coumarate:CoA ligase

Figure 4 - Steady State Model of Cinnamaldehyde Production from an L-Phenylalanine Precursor using 10 times the concentration of the wild type isoform of 4-coumarate:CoA ligase

Figure 5 - Modeling Source Code

Table 1 - Enzyme Modeling Kinetics and Environmental Conditions

References

1. Mulquiney, P.J., Kuchel, P.W. Modeling Metabolism with Mathematica. CRC Press: 2003.
2. Schneider, K., Hovel, K., Witzel, K., Hamberger, B., Schombur, D., Kombrink, E., Stuible, H.P. 2003. The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase. PNAS. 100, 8601-8606.

3. Stuible, H.P., Buttner, D., Ehlting, J., Hahlbrock, K., Kombrink, E. 2000. Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes. FEBS Letters. 467, 117-122.

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