W0326

Structural Analysis of the Type I Nitroreductase From Enterobacter Cloacae Reveals the Effects of Co-Factor Reduction and Substrate Binding. Chad A. Haynes, Ronald L. Koder, Anne-Frances Miller, David W. Rodgers, Molecular and Cellular Biochemistry, Univ. of Kentucky, Lexinton, KY 40536.

The type I nitroreductase (NR) enzyme from E. cloacae is responsible for the NAD(P)H dependent reduction of nitroaromatic, quinone, and riboflavin compounds. NR utilizes a tightly bound flavin mononucleotide (FMN) molecule as the enzymatic co-factor. The enzyme displays broad substrate specificity but reacts most readily with the explosive compounds tri-nitrotoluene and di-nitrotoluene. We have pursued both biochemical and structural studies of the enzyme in order to gain insight into the details of the enzymatic mechanism, including both substrate binding and flavin chemistry. In these studies, we have determined the crystal structures of oxidized NR bound to two inhibitors, acetate and benzoate, as well as the substrate analogs nicotinic acid adenine dinucleotide (NAAD) and para-nitrobenzoic acid (p-NBA). Furthermore, we have determined the crystal structure of the two-electron reduced enzyme.

The enzyme is a homodimer that adopts an a+b fold and binds two flavin mononucleotide molecules at the dimer interface, creating two independent active sites. The geometry of the flavin ring system is affected by its redox state; the oxidized flavin is nonplanar, with an overall bend angle of 16∞; the bend angle increases to 25∞ in the reduced flavin. Since free, oxidized flavin is planar in solution, the bend induced by binding to the apo enzyme may thermodynamically encourage formation of the reduced flavin, which is bent when free in solution. The conformation imposed on the bound oxidized flavin may disfavor formation of the one-electron reduced or semi-quinone state of the flavin, since it too, like the oxidized form, is planar in solution.

Also, comparison between the two different competitive inhibitor complexes shows that a portion of helix H6 can flex to accommodate the differently sized inhibitors suggesting a mechanism for binding a variety of substrates of differing sizes.

We will also report on the binding details of the NADH analog, NAAD, and the second half reaction substrate p-NBA. General features including positioning of the small molecules within the active site along with the concomitant expansion of the active site will be presented.