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

Abstract

In the biosynthesis of the tRNA-inserted nucleoside queuosine, the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine (preQ 1 ), 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 0 which undergoes reduction to preQ 1 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 E. coli, the conserved Glu 89 and Phe 228 residues together with a mobile structural element comprising the catalytic Cys 190 form a substratebinding pocket that secludes the bound preQ 0 completely from solvent. We show here that residue substitutions (E89A, E89L, F228A) targeted at opening up the binding pocket weakened preQ 0 binding at the preadduct stage, by up to +10 kJ/mol, and profoundly affected on catalysis. Unlike wildtype enzyme, the QueF variants, including L191A and I192A, were no longer selective for preQ 1 formation. The E89A, E89L and F228A variants performed primarily (≥ 90%) a two-electron reduction of preQ 0 , releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product. The preQ 0 reduction by L191A and I192A gave preQ 1 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 0 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-toamine conversion by this class of enzymes.

Unexpected NADPH Hydratase Activity in the Nitrile Reductase QueF from Escherichia coli

“…The proposed mechanism of QueF catalysis involves a thioimidate covalent enzyme‐preQ 0 adduct that undergoes reduction by NADPH in two catalytic steps via an imine intermediate (Scheme ) . The QueF active site consists of a cysteine/aspartate dyad of residues that operate in a functionally interconnected manner in covalent enzyme catalysis, as shown in Scheme .…”

“…Site‐directed mutagenesis and enzyme preparation : Mutagenesis to substitute Glu89 with Leu (E89L), Cys190 with Ala (C190A) and Ser (C190S), and Asp197 with Ala (D197A) or His (D197H) was reported in earlier studies of ecQueF . Genes encoding the double variants (C190A/D197A, E89L/D197A, E89Q/D197A) were obtained from Genscript (Piscataway, NJ, USA).…”

“…All mutations were verified by gene sequencing. The ecQueF variants were obtained as N‐terminally His‐tagged proteins through expression in E. coli BL21‐DE3 as described previously . All enzymes were purified by use of a reported two‐step procedure consisting of immobilized metal ion affinity chromatography and gel filtration .…”

Interplay of nucleophilic catalysis with proton transfer in the nitrile reductase QueF from Escherichia coli

“…12,[22][23][24][25][26] The natural reaction of QueF is conversion of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine (preQ 1 ) by NADPH (Scheme 1B). [17][18][19]21,27,28 QueF is a bacterial enzyme from the biosynthetic pathway for the modified nucleoside queuosine (Q). 12,22,29 Inserted into tRNA as preQ 1 and further converted to Q, queuosine modulates the codon-anticodon binding efficiency for decoding NAC/U codons to Asn, Asp, His, and Tyr.…”

“…The core characteristics of this mechanism are well supported by enzyme crystal structures and biochemical evidence. 17,18,21,27,28 Therefore, the thioimidate adduct is formed from a non-covalent QueF-preQ 0 complex that secludes the nitrile substrate completely from the solvent (Schemes 1B and 2). [17][18][19][20] The covalent intermediate is converted to preQ 1 in two NADPH dependent reduction steps via an imine.…”