Expression and characterization of the nitrile reductase queF from E. coli

Moeller, Kathrin; Nguyen, Giang-Son; Hollmann, Frank; Hanefeld, Ulf

doi:10.1016/j.enzmictec.2012.12.003

Cited by 21 publications

(19 citation statements)

References 15 publications

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“…QueF enzymes from Bacillus subtilis [1,11], Vibrio cholera [12], Geobacillus kaustophilus [13], and Escherichia coli [14,15] have been biochemically characterized, and a number of mechanistic investigations have been reported [11,16,17,18]. The reaction begins with the binding of preQ 0 and its reaction with the thiol of an active site cysteine (Cys55 in the B. subtilis enzyme) [11,16] to form a covalent thioimide intermediate (Figure 2, I).…”

Section: Introductionmentioning

confidence: 99%

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide

et al. 2017

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QueF enzymes catalyze the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the biosynthetic pathway to the tRNA modified nucleoside queuosine. The QueF-catalyzed reaction includes formation of a covalent thioimide intermediate with a conserved active site cysteine that is prone to oxidation in vivo. Here, we report the crystal structure of a mutant of Bacillus subtilis QueF, which reveals an unanticipated intramolecular disulfide formed between the catalytic Cys55 and a conserved Cys99 located near the active site. This structure is more symmetric than the substrate-bound structure and exhibits major rearrangement of the loops responsible for substrate binding. Mutation of Cys99 to Ala/Ser does not compromise enzyme activity, indicating that the disulfide does not play a catalytic role. Peroxide-induced inactivation of the wild-type enzyme is reversible with thioredoxin, while such inactivation of the Cys99Ala/Ser mutants is irreversible, consistent with protection of Cys55 from irreversible oxidation by disulfide formation with Cys99. Conservation of the cysteine pair, and the reported in vivo interaction of QueF with the thioredoxin-like hydroperoxide reductase AhpC in Escherichia coli suggest that regulation by the thioredoxin disulfide-thiol exchange system may constitute a general mechanism for protection of QueF from oxidative stress in vivo.

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Section: Introductionmentioning

confidence: 99%

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide

et al. 2017

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“…[8] The cloning, overexpression, and biochemical characterization of nitrile reductase QueF (also termed 7-cyano-7-deazaguanine reductase) from various bacterial sources, such as Escherichia coli, Bacillus subtilis, Vibrio cholerae, and Geobacillus kaustophilus have been reported. [9][10][11][12] Based on their amino acid sequences, two structural subfamilies of nitrile reductases QueF are known: the YqcD subfamily (type II) are two-domain proteins, to which the enzymes from V. cholerae and E. coli belong, whereas the YkvM subfamily (type I) includes single-domain proteins, such as the enzyme from B. subtilis. [13] Scheme 1 Nitrile Reduction by QueF in the Biosynthesis of Queuosine in Bacteria [8] Mechanism A catalytic mechanism for nitrile reductase QueF has been proposed based on crystal structure analysis combined with molecular simulations (Scheme 2).…”

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confidence: 99%

“…In order to examine the potential of enzymatic nitrile reduction, wild-type and mutant enzymes of nitrile reductase from E. coli as well as the thermophile G. kaustophilus were employed using a broad range of structurally diverse nonnatural substrates. [10][11][12] The enzyme variants were prepared by site-directed mutagenesis based on homology models. [11,14] The activities (initial rates) were analyzed by spectrophotometric monitoring of NADPH depletion (at 340 nm) and HPLC/MS measurements to determine conversions, which were normalized to the preQ 0 reductase activity.…”

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confidence: 99%

“…[11,14] The activities (initial rates) were analyzed by spectrophotometric monitoring of NADPH depletion (at 340 nm) and HPLC/MS measurements to determine conversions, which were normalized to the preQ 0 reductase activity. [11,12] Unfortunately, the substrate scope of the nitrile reductases turned out to be very narrow which limits their applicability in biocatalysis (Scheme 3).…”

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confidence: 99%

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3.10 Emerging Enzymes

Glueck,

Hammer,

Hauer

et al. 2015

Biocatalysis in Organic Synthesis 3

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The broad application of enzymes in industry is still impeded by the limited repertoire of available enzymes. New applications of biocatalysts in a sustainable bio-based economy, especially for employment in bioremediation, bioenergy, and bioproducts, appear to be the driving force behind the increased prevalence of novel enzymatic catalysts. Advances in synthetic biology, enzyme engineering, de novo enzyme (re)design, exploitation of enzyme promiscuity, and DNA sequencing technologies show great potential to facilitate the development of more efficient enzymes. Combined efforts would have the following benefits: an increased range of biocatalysts with different catalytic activities would be available and more biocatalysts with improved characteristics suitable for (bio)synthetic chemistry approaches would be discovered. This chapter demonstrates the potential of enzymes that have been investigated recently, but not yet fully exploited, for applications in chemical synthesis: nitrile reductases, sulfatases, phenol-coupling monooxygenases, squalene hopene cyclases, and aldoxime dehydratases. Examples of emerging enzymes and microorganisms that have been studied over the last few years and that have acquired scientific as well as industrial interest are described to illustrate the synthetic potential of these biocatalysts.The screening reactions were run in UV star 96-well plates using recombinant His-tagged enzymes purified via nickel affinity chromatography. The following conditions were applied: nitrile reductase (45 ìM, purity >85%) in buffer [100 mM Tris, 50 mM KCl, 1.15 mM 3.10.

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“…The core Tfold domain consists of a ββααββ arrangement of secondary structure (4,5,9). The known QueF enzymes are divided into unimodular (e.g., QueF from Bacillus subtilis; bsQueF) (3,5,19,20) and bimodular groups (e.g., QueF from Escherichia coli; ecQueF) (4,7,(21)(22)(23)(24), depending on whether they comprise subunits containing only a single Tfold domain or two T-fold domains in tandem repeat, respectively. Both types of QueF have their active sites located at the structural interface of two T-fold domains.…”

Section: Introductionmentioning

confidence: 99%

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

Jung

Nidetzky

2018

Journal of Biological Chemistry

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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.

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Expression and characterization of the nitrile reductase queF from E. coli

Cited by 21 publications

References 15 publications

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide

Protection of the Queuosine Biosynthesis Enzyme QueF from Irreversible Oxidation by a Conserved Intramolecular Disulfide

3.10 Emerging Enzymes

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

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