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

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish
Pages (from-to)3720-3733
Number of pages14
JournalJournal of Biological Chemistry
Volume293
Issue number10
DOIs
Publication statusPublished - 1 Jan 2018

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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