Conclusion

NemA and Xen B are capable of catalysing the conversion of TNT to various products using NADH and FMN as cofactors. The binding affinity of each protein for this substrate (the Michealis Menten constant, Km) and the maximum reaction velocity (Vmax), were determined and are comparable to the published values; shown in figure 1. NemA and XenB are therefore suitable enzymes for use in our system and have been shown to function at the physiologically relevant pH of 7.

	NemA Vmax (TNT)	XenB Vmax (TNT)	NemA Vmax (Nitroglycerin)	XenB Vmax (Nitroglycerin)	NemA Km (TNT)	XenB Km (TNT)	NemA Km (Nitroglycerin)	XenB Km (Nitroglycerin)
Experimental Results
Published Values			8				15

Figure 1

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Abstract

NemA and XenB are two proteins that the iGEM Exeter team propose will allow E.coli to degrade TNT, at concentrations above those normally toxic to the cell. Among many others proposed, NemA catalyses the reaction shown in figure 2.

Figure 2

Various hydroxylamino derivatives may be produced, as well as ammonium ions which could be used as a nitrogen source by the E.coli for growth. The following experimental account describes the protocol used to confirm the substrate target of NemA and XenB and analyse the respective kinetic capabilities of these enzymes.

Results

Choice of analytical procedure

NADH is a cofactor in the conversion of TNT to X. This opens up the possibility to simply measuring the catalytic rate of TNT degradation by following change in the absorbance at 340nm. However, very early in the process we realised that TNT also produces a significant absorbance at 340nm, a fact that would complicate our analytical procedure. We therefore chose to use High Performance Liquid Chromatography (HPLC) analysis. As can be seen (Fig X), HPLC separates TNT and NAD by elution time meaning that the overlap in absorbance values are not an issue.

TNT Standard Curve

A standard curve of TNT concentration was determined first. Integration of the area below the absorbance peak of TNT at the concentrations described, resulted in the standard curve shown in figure 3. For further details see materials and methods.

Figure 3: Standard curve demonstrating HPLC peak area response to different concentrations of TNT

Determination of NemA activity

Purified NemA protein was assayed for its ability to degrade TNT at a range of concentrations: from 0 mM TNT up to 4.4 mM TNT. The reaction is a stopped enzyme assay. In this system the reaction is started by the addition of NemA to a reaction mix containing TNT, FMN and NADH. The reaction is stopped by the addition of hydrochloroacetic acid at predetermined intervals and the concentration of TNT is then assayed using the HPLC. These data are shown in figure 4. Full details can be seen in the materials and methods.

Figure 4

Figure 5 shows NemA reaction kinetics as a function of initial velocity, Vi, against initial TNT concentration (derived from taking a tangent at the steepest section of each series in figure 4). From this it can be concluded that the Vmax is...

Figure 5: NemA reaction kinetics as a function of initial velocity, Vi, against initial TNT concentration

The data can either be plotted as a Lineweaver-Burke plot (figure 6), or as a Hanes plot (figure 7) to more accurately determine the Km values. From these it can be concluded that the Km of NemA for TNT substrate is...

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