Evidence of a sequestered imine intermediate during reduction of nitrile to amine by the nitrile reductase QueF from Escherichia coli

In the biosynthesis of the tRNA-inserted nucleoside queuosine, the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ₀) to 7-aminomethyl-7-deaza-guanine (preQ₁), a biologically unique four-electron reduction of a nitrile to an amine. The QueF mechanism involves a covalent thioimide adduct between the enzyme and preQ₀ that undergoes reduction to preQ₁ in two NADPH-dependent steps, presumably via an imine intermediate. Protecting a labile imine from interception by water is fundamental to QueF catalysis for proper enzyme function. In the QueF from Escherichia coli, the conserved Glu⁸⁹ and Phe²²⁸ residues together with a mobile structural element composing the catalytic Cys¹⁹⁰ form a substrate-binding pocket that secludes the bound preQ₀ completely from solvent. We show here that residue substitutions (E89A, E89L, and F228A) targeted at opening up the binding pocket weakened preQ₀ binding at the preadduct stage by up to 10 kJ/mol and profoundly affected catalysis. Unlike wildtype enzyme, the QueF variants, including L191A and I192A, were no longer selective for preQ₁ formation. The E89A, E89L, and F228A variants performed primarily (>90%) a two-electron reduction of preQ₀, releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product. The preQ₀ reduction by L191A and I192A gave preQ₁ and 7-formyl-7-deazaguanine at a 4:1 and 1:1 ratio, respectively. The proportion of 7-formyl-7-deazagua-nine in total product increased with increasing substrate concentration, suggesting a role for preQ₀ in a competitor-induced release of the imine intermediate. Collectively, these results provide direct evidence for the intermediacy of an imine in the QueF-catalyzed reaction. They reveal determinants of QueF structure required for imine sequestration and hence for a complete nitrile-to-amine conversion by this class of enzymes.