In the biosynthesis of the tRNA-inserted nucleoside queuosine， the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ) to 7-aminomethyl-7-deazaguanine (preQ)， a biologically unique four-electron reduction of a nitrile to an amine. The QueF mechanism involves a covalent thioimide adduct between the enzyme and preQ that undergoes reduction to preQ 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 ， the conserved Glu and Phe residues together with a mobile structural element composing the catalytic Cys form a substrate-binding pocket t... More
In the biosynthesis of the tRNA-inserted nucleoside queuosine， the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ) to 7-aminomethyl-7-deazaguanine (preQ)， a biologically unique four-electron reduction of a nitrile to an amine. The QueF mechanism involves a covalent thioimide adduct between the enzyme and preQ that undergoes reduction to preQ 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 ， the conserved Glu and Phe residues together with a mobile structural element composing the catalytic Cys form a substrate-binding pocket that secludes the bound preQ completely from solvent. We show here that residue substitutions (E89A， E89L， and F228A) targeted at opening up the binding pocket weakened preQ 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 preQ formation. The E89A， E89L， and F228A variants performed primarily (≥90%) a two-electron reduction of preQ， releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product. The preQ reduction by L191A and I192A gave preQ and 7-formyl-7-deazaguanine at a 4:1 and 1:1 ratio， respectively. The proportion of 7-formyl-7-deazaguanine in total product increased with increasing substrate concentration， suggesting a role for preQ 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.
