Isotope Effects and Kinetic Mechanism of Enterobacter cloacae Nitroreductase

Abstract

Nitroreductase from E. cloacae (NR) is a member of a large family of homologues represented in all branches of the tree of life. However the physiological roles of many of these enzymes remain unknown. NR has distinguished itself on the basis the diverse substrates it can reduce (1). This might be an evolved characteristic suiting NR for a role in metabolism of diverse occasional toxins. While there are numerous studies of determinants of substrate specificity, we know less about mechanisms by which enzymes can be inclusive. Therefore, we have characterized the fundamental steps of NR's mechanism including substrate binding, electron transfer and proton transfer, using a slow substrate. We find that NR reduces para-nitrobenzoic acid (p-NBA) via a simple mechanism rate-limited by the chemical step in which the nitro group is reduced (2). Thus for this substrate, NR dispenses with gating steps that can enforce substrate specificity. Our data demonstrate that substrate reduction is accomplished by rate-contributing hydride transfer from the flavin cofactor coupled to rate-contributing proton transfer from solvent. This is consistent with our crystal structures, which reveal a spacious solvent-exposed active site bounded by a helix that moves to accommodate binding of substrate analogs (3). Because it is able to reduce herbicides, pesticides and inexpensive nitroaromatic starting materials, NR has potential utility in bioremediation and in production of intermediates needed for synthesis of pharmaceuticals.