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Figure 1: HSQC of NR at 37 oC (red) compared with the HSQC of NR at 4 oC (black).

Figure 2: Mechanisms of the two half-reactions of NR. Crystallographic models were solved with the NADH analog nicotinic acid adenine dinucleotide (NAAD) bound to oxidized NR, or substrate pNBA bound to oxidized NR. Kinetic studies demonstrate hydride transfers in both steps and the applicable Hs are in teal/mint green in the structural cartoons, and red in the chemical structures.

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

Dynamics related to promiscuity?

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 to perform new tasks by increasing protein flexibility. We are studying a nitroreductase (NR) which can reduce nitrated aromatics as well as diverse herbicides, pesticides and antibiotics [22], cloned from Enterobacter cloacae growing in a weapons storage facility. This reactivity is presumed to represent a recent adaptation. NR displays pronounced, widely-distributed intermediate time-scale dynamics at room temperature that is exacerbated at low temperature. It also displays a significant conformational change upon substrate analog binding, based on X-ray crystallography [39]. Therefore we propose that catalytic turnover and/or substrate binding involves dynamics.

Mechanistic underpinnings of promiscuity.

Many familiar enzymes undergo conformational changes in the course of turnover, and the different conformational states can enable the protein to 'test' the substrate and exercise selectivity at different points in the catalytic cycle. These events can manifest themselves via distinct kinetic phases and intermediates detectable in pre-steady-state kinetic studies. Therefore we conducted stopped-flow studies of both of NR's two half reactions (reduction of the flavin by NADH, reoxidation of the flavin by para-nitrobenzoic acid, pNBA) [82,88]. pNBA was chosen because it is a slow substrate and therefore provides a good chance of probing the chemical step(s) and detecting intermediates. It is also a substrate for which a crystallographic model could be solved [88].
            Our kinetic studies reveal a very simple mechanism consistent with absence of selective gating steps [82]. Isotope dependencies moreover demonstrate that both half reactions proceed via a hydride transfer [88]. This represents a change in binding mode upon flavin reduction. Such has also been demonstrated for the E. coli homologue as well as pentaerythritol tetranitrate reductase. Our kinetic studies thus overturn computational mechanisms and provide a valuable adjunct to crystallographic studies by arbitrating between two possible conformations of the substrate. Thus our mechanistically-informed crystal structure provides a basis for future modelling and mutagenesis studies aimed at modifying the substrate repertoire of NR to enable greater utility in bioremediation applications and synthesis of valuable precursors for synthesis of pharmaceuticals.

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Updated: Oct. 2017                                                                                               

Copyright 2017 A.-F. Miller     

Comments: A.-F. Miller