Figure 2: HSQC of NR at 37 °C (red) compared with the HSQC of NR at 4 °C (black).

Figure 3: Residues whose backbone amide temp. dependence could be followed by NMR, color-coded according to the lowest temp. at which they retained a unique persistant conformation.

Significance of protein dynamics to the broad substrate repertoire of nitroreductase.

Protein dynamics is widely held to be essential for catalytic activity however the nature and magnitude of the contribution remains poorly understood. We propose that increased protein dynamics provides a rapid means of expanding the substrate specificity of an enzyme without changing the nature of the reactivity. Thus existing enzymes could be recruited in nature, and in practice, to perform new tasks by increasing protein flexibility. We are studying nitroreductase (NR), which was cloned from bacteria growing in a weapons storage facility, and can reduce nitrated aromatics rapidly. This reactivity is presumed to represent a recent adaptation, whereas the enzymes’ ability to reduce quinones is believed to represent the ancestral physiological role of this enzyme. NR displays pronounced, widely-distributed intermediate time-scale dynamics at room temperature that is suppressed at 2 °C, and NR also undergoes a significant conformational change upon substrate analog binding, based on X-ray crystallography. Therefore, we propose that catalytic turnover involves protein dynamics. We will use NMR measurements of 15N and 2H nuclear spin relaxation rates to evaluate the timescale vs. magnitude of motions throughout NR and a homologous enzyme from an extreme thermophile. For NR, we will determine whether the temperature profile for NMR-detected dynamics parallels that for catalytic activity (it does not correlate with protein denaturation). We will do the same for the hyperthermophile to learn whether its higher temperature optimum for activity is associated with a similarly shifted profile for protein dynamics. If so, we will be able to identify a single temperature corresponding to a low-temperature for the thermophile but a high one for NR, and perform a detailed characterization of dynamics to identify differences between the two enzymes that would therefore be related to any differences in their temperature dependencies of activity.

Thus, these studies will test a relationship between dynamics and activity, per se. We will test our hypothesis that dynamics expands the substrate specificity by comparing the temperature profile of activity with respect to a quinone substrate (one for which the enzyme was presumably optimized) with the profile for a nitrated aromatic (a presumed recent addition to NR’s repertoire). Thus, we will determine whether or not increased dynamics relaxes NR’s substrate specificity.

Specific hypotheses arising from these characterizations will be tested by mutating implicated residues, especially residues that distinguish NADOX from NR and studies involving the use of chaotropes.

 

last updated: May, 2008 comments: send email to Cungen 
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