Recombinant Desulfovibrio vulgaris NADPH-dependent 7-cyano-7-deazaguanine reductase (queF)

What is QueF and what role does it play in queuosine biosynthesis?

QueF is an enzyme that catalyzes a critical late step in the biosynthesis of queuosine, a modified nucleoside found in the wobble position of certain tRNAs. Specifically, QueF catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ₀) to 7-aminomethyl-7-deazaguanine (preQ₁), which is subsequently inserted into tRNA by the enzyme tRNA-guanine transglycosylase (TGT) . This reduction represents the only known biological pathway that includes the complete reduction of a nitrile bond to a primary amine, making QueF particularly noteworthy from a mechanistic perspective . The queuosine biosynthetic pathway begins with GTP or a related metabolite and proceeds through several intermediates, with QueF catalyzing the penultimate step before the modified base is incorporated into tRNA .

What is the catalytic function of QueF?

QueF functions as an NADPH-dependent oxidoreductase that catalyzes the reduction of the nitrile group (CN) in 7-cyano-7-deazaguanine (preQ₀) to a primary amine (H₂C-NH₂), forming 7-aminomethyl-7-deazaguanine (preQ₁) . This remarkable transformation involves the transfer of four protons and electrons to the substrate . The initial step of the catalytic process involves the formation of a covalent thioimide intermediate between the substrate and a conserved cysteine residue in the active site . Subsequent steps involve NADPH-dependent reduction of this intermediate, with additional conserved residues facilitating proton transfer to complete the reaction. The reduction can be monitored through a continuous UV-based assay tracking NADPH oxidation, and the product formation can be confirmed through HPLC analysis and NMR spectroscopy .

What structural characteristics define QueF enzymes?

QueF enzymes belong to the tunneling fold (T-fold) superfamily and are categorized into two structural subfamilies:

• Unimodular QueF (e.g., B. subtilis QueF): These are composed of subunits with a single T-fold domain and form homodecamers consisting of two head-to-head facing pentamers arranged in a cyclic formation, creating a central tunnel. Each homodecamer contains 10 active sites located at the intersubunit interfaces .

• Bimodular QueF (e.g., V. cholerae and E. coli QueF): These contain subunits with two weakly homologous tandem T-fold domains and form homodimers with two active sites located at the interfaces between the two T-fold domains within each monomeric subunit .

Both subfamilies share a conserved QueF motif E(S/L)K(S/A)hK(L/Y)(Y/F/W) (where h is a hydrophobic residue), which contains residues responsible for NADPH binding . This motif is embedded in a helix flanking the active site. The active sites in both subfamilies contain an invariant glutamate residue that anchors the preQ₀ substrate and an invariant cysteine residue that forms the thioimide intermediate with preQ₀ .

What cofactors are required for QueF activity?

QueF requires NADPH as its primary cofactor for catalytic activity. NADPH serves as the electron donor in the reduction of the nitrile group to a primary amine . Through kinetic analysis, the Km for NADPH has been determined to be approximately 36 μM, which is consistent with the Km values observed for other bacterial NADPH-dependent oxidoreductases . The conserved QueF motif in the enzyme structure contains residues specifically responsible for NADPH binding . While testing various redox cofactors, research has demonstrated that QueF activity is dependent on NADPH, with the rate of NADPH oxidation proportional to enzyme and substrate concentrations, confirming QueF's role as the catalyst in the redox reaction between preQ₀ and NADPH .

What are the key residues involved in the catalytic mechanism of QueF?

Several highly conserved residues play crucial roles in the catalytic mechanism of QueF enzymes:

In unimodular QueF, these conserved elements are contained within the same T-fold domain, whereas in bimodular QueF, the QueF motif and the active site Cys and Glu are separated in the two domains and join together in the tertiary structure to form an interdomain active site . Numerical simulations have provided insight into the mechanism by which this enzyme affects the transfer of four protons and electrons to the substrate, confirming that the initial step involves the formation of a covalent adduct with the catalytic cysteine residue .

How does oxidative stress affect QueF activity and structure?

The catalytic cysteine of QueF has been shown to be prone to oxidation in vivo in the proteomes of several bacterial species when exposed to oxidative stress . In B. subtilis QueF, crystallographic evidence reveals that under oxidative conditions, an intramolecular disulfide bond forms between the catalytic Cys55 and a conserved Cys99 located in a helix lining the active site . This disulfide formation causes significant structural rearrangements, particularly in the loops responsible for substrate binding, resulting in a more symmetric structure compared to the substrate-bound form .

The following table summarizes the properties of the protective disulfide in QueF:

Mutational studies demonstrate that Cys99 is not essential for catalytic activity (Cys99Ala and Cys99Ser mutants retain full activity), but its presence protects the enzyme from irreversible oxidative damage . When the enzyme is oxidized, activity can be restored by treatment with thioredoxin, which reduces the disulfide bond . This protection mechanism appears to be biologically relevant, as QueF has been shown to interact in vivo with the thioredoxin-like alkyl hydroperoxide reductase AhpC in E. coli .

What experimental approaches can be used to study QueF catalytic mechanism?

Multiple experimental strategies are valuable for investigating the catalytic mechanism of QueF:

• Structural Analysis

  • X-ray crystallography to determine enzyme structure in different states (apo, substrate-bound, intermediate-bound)

  • Solution-phase structural techniques (SAXS, NMR) to assess conformational dynamics

• Kinetic Analyses

  • Continuous UV-based assays to monitor NADPH oxidation during catalysis

  • HPLC analysis to confirm product formation and quantify reaction progress

  • Pre-steady-state kinetics to capture transient intermediates

• Spectroscopic Methods

  • UV-visible spectroscopy for monitoring cofactor states and reaction progress

  • Mössbauer and EPR spectroscopy for analyzing metal centers if present

  • NMR spectroscopy for confirming product identity and structural changes

• Mutagenesis Studies

  • Site-directed mutagenesis of conserved residues (e.g., Cys99Ala, Cys99Ser) to assess functional roles

  • Alanine-scanning mutagenesis of active site residues to map catalytic contributions

• Computational Approaches

  • Numerical simulations to model proton and electron transfer mechanisms

  • Molecular dynamics to analyze protein conformational changes

  • Quantum mechanical calculations to determine transition state energetics

• Redox Biology Techniques

  • Thioredoxin-dependent reactivation assays to assess oxidative regulation

  • Analysis of disulfide bond formation under various redox conditions

This multidisciplinary approach has revealed important insights into QueF's unique nitrile reductase activity and protection mechanisms against oxidative stress.

How can recombinant D. vulgaris QueF be expressed and purified for biochemical studies?

While the search results don't specifically address D. vulgaris QueF expression, insights can be gained from the recombinant expression of other D. vulgaris proteins and QueF from other organisms:

The gene encoding D. vulgaris QueF would first need to be cloned into an appropriate expression vector for E. coli. Based on experience with other D. vulgaris proteins, such as rubrerythrin, the recombinant protein may be expressed in an insoluble form and potentially deficient in cofactors . For rubrerythrin, researchers successfully incorporated iron in vitro by:

• Dissolving the insoluble protein in 3 M guanidinium chloride

• Adding Fe(II) anaerobically

• Diluting the denaturant to allow refolding

A similar approach could be adapted for QueF, though without the iron incorporation step. Alternatively, expression conditions could be optimized to improve solubility:

• Lowering the induction temperature (e.g., 18-20°C)

• Reducing inducer concentration

• Co-expressing with molecular chaperones

• Using specialized E. coli strains designed for difficult proteins

Purification would likely involve:

• Cell lysis under conditions that maintain enzyme activity

• Initial capture by affinity chromatography (if a fusion tag is used)

• Secondary purification by ion exchange and/or size exclusion chromatography

• Analysis of purity by SDS-PAGE and activity assays

For activity assays, a continuous UV-based assay monitoring NADPH oxidation would be appropriate, followed by HPLC confirmation of preQ₁ production .

What are the kinetic parameters of QueF and how do they compare across species?

While specific kinetic parameters for D. vulgaris QueF are not provided in the search results, known parameters for QueF enzymes from other organisms provide valuable comparison points:

These parameters indicate that QueF has evolved for high substrate specificity (low Km for preQ₀) rather than rapid turnover (relatively low kcat). The high affinity for preQ₀ is consistent with its role in a biosynthetic pathway for a modified nucleoside, where substrate concentrations are likely to be low in vivo.

To determine the specific kinetic parameters for D. vulgaris QueF, researchers would need to:

• Express and purify the recombinant enzyme

• Perform steady-state kinetic analyses with varying concentrations of both substrates

• Analyze the data using appropriate enzyme kinetic models (e.g., Michaelis-Menten, bi-substrate kinetic models)

How can site-directed mutagenesis be used to investigate QueF mechanism?

Site-directed mutagenesis represents a powerful approach for investigating the roles of specific residues in the QueF catalytic mechanism. Based on insights from studies of B. subtilis and V. cholerae QueF, several targeted mutations would be informative:

• Catalytic Cysteine Mutations

  • Mutation of the catalytic cysteine (equivalent to Cys55 in B. subtilis or Cys194 in V. cholerae) to serine or alanine

  • Expected outcome: Loss of thioimide intermediate formation and catalytic activity

  • Analytical methods: Activity assays, mass spectrometry to confirm absence of covalent intermediate

• Substrate Binding Residue Mutations

  • Mutation of the conserved glutamate (equivalent to Glu97 in B. subtilis or Glu234 in V. cholerae) that anchors preQ₀

  • Expected outcome: Increased Km for preQ₀, decreased catalytic efficiency

  • Analytical methods: Steady-state kinetics, isothermal titration calorimetry for binding affinity

• Protective Cysteine Mutations

  • Mutation of the secondary cysteine involved in disulfide formation (equivalent to Cys99 in B. subtilis)

  • Expected outcome: Maintained catalytic activity but increased susceptibility to irreversible oxidation

  • Analytical methods: Activity assays before and after oxidative treatment, with and without thioredoxin reduction

• NADPH Binding Residue Mutations

  • Alanine substitutions of residues in the QueF motif

  • Expected outcome: Altered NADPH binding affinity and possibly catalytic rate

  • Analytical methods: Steady-state kinetics with varying NADPH concentrations, fluorescence-based binding assays

• Proton Transfer Residue Mutations

  • Mutations of residues implicated in proton delivery (equivalents to His233 and Asp102 in V. cholerae)

  • Expected outcome: Altered reaction rate, possible accumulation of intermediates

  • Analytical methods: pH-dependent kinetics, spectroscopic analysis of reaction intermediates

The results from these mutational studies would provide crucial insights into the catalytic mechanism, substrate specificity, and oxidative regulation of D. vulgaris QueF, contributing to a more comprehensive understanding of this unique nitrile reductase.