Recombinant Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni NADPH-dependent 7-cyano-7-deazaguanine reductase (queF)

What is queF and what reaction does it catalyze in Leptospira interrogans?

QueF (NADPH-dependent 7-cyano-7-deazaguanine reductase) catalyzes the unprecedented four-electron reduction of a nitrile group to a primary amine, specifically converting 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1) in the queuosine biosynthetic pathway. This represents the only known biological reaction of this kind and represents a fifth class of enzymes responsible for nitrile metabolism . In Leptospira interrogans serogroup Icterohaemorrhagiae serovar copenhageni, queF (UniProt ID: Q72RB6) consists of 133 amino acids and functions in the tRNA modification pathway essential for bacterial protein translation .

How does queF function in the biosynthesis of queuosine?

QueF functions in a late step of queuosine biosynthesis. Queuosine is a 7-deazaguanine modified nucleoside found in tRNA GUN of Bacteria and Eukarya. The biosynthetic pathway begins with GTP and proceeds through several intermediates. QueF specifically catalyzes the conversion of preQ0 to preQ1, which is subsequently inserted into tRNA by the enzyme TGT (tRNA-guanine transglycosylase). This conversion is essential for the formation of the fully modified queuosine base . The preQ1 intermediate is specifically recognized by TGT with a Km of approximately 0.39 μM, highlighting the coordinated affinity between consecutive enzymes in this pathway .

What expression systems are commonly used for producing recombinant L. interrogans queF?

E. coli is the predominant expression system used for recombinant production of L. interrogans queF. Typically, the queF gene is cloned into expression vectors with suitable affinity tags (most commonly His-tag) to facilitate purification. For example, recombinant L. interrogans serogroup Icterohaemorrhagiae serovar copenhageni queF has been successfully expressed in E. coli systems to produce the full-length protein (133 amino acids) with greater than 85% purity as determined by SDS-PAGE . The recombinant protein is typically provided as a lyophilized powder and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with glycerol added to a final concentration of 5-50% for long-term storage .

What are the detailed steps of the catalytic mechanism of queF?

The catalytic mechanism of queF has been elucidated through QM/MM calculations and experimental studies, revealing four major stages:

• Thioimidate formation: Formation of a C–S covalent bond between the substrate preQ0 and the catalytic cysteine residue (Cys190 in E. coli, Cys55 in B. subtilis)

• First hydride transfer: NADPH transfers its hydride to the substrate to generate a thiohemiaminal intermediate

• C-S bond cleavage: The C–S covalent bond is cleaved to generate an imine intermediate

• Second hydride transfer: A second hydride from NADPH is transferred to the imine intermediate to generate the final amine product (preQ1)

The rate-limiting step in this process is the second hydride transfer, with a calculated free energy barrier of 20.8 kcal/mol . Both hydride transfer steps specifically involve the 4-pro-R-hydrogen from NADPH . Kinetic isotope effect studies reveal a large primary deuterium kinetic isotope effect of 3.3 on the covalent thioimide reduction and a smaller kinetic isotope effect of 1.8 on the imine reduction to preQ1 .

How can researchers accurately measure and characterize the enzymatic activity of queF?

Several complementary methods have been established for analyzing queF activity:

Continuous UV-based assay: This approach monitors NADPH oxidation at 340 nm, which directly correlates with enzyme activity. The rate of NADPH consumption depends on enzyme and substrate concentrations, providing a real-time measurement of queF catalytic activity .

HPLC analysis: HPLC can be used to confirm product formation by identifying the appearance of preQ1. In previous studies, a new peak appeared at approximately 14 minutes that coeluted with authentic preQ1 and had an identical UV-visible spectrum. This peak can be isolated and further analyzed by proton NMR to confirm product identity .

Fluorescence assay: A fluorescence-based method optimized to follow the formation of NADP+ as an alkaline degradation product provides another approach for determining kinetic constants. This method has been used to determine Km values for preQ0 as low as 0.237 ± 0.045 μM .

For accurate kinetic characterization, researchers should determine the following parameters:

• Km for NADPH (typically 19-36 μM in bacterial queF enzymes)

• Km for preQ0 (typically < 1 μM)

• kcat (approximately 0.6-0.7 min-1 in characterized queF enzymes)

What techniques can be used to probe the structural features critical for queF function?

Several complementary approaches have proven effective for investigating queF structure-function relationships:

Site-directed mutagenesis: Targeting conserved residues, particularly the catalytic cysteine (Cys190 in E. coli, Cys55 in B. subtilis), which has been shown to be essential for nucleophilic attack on the nitrile substrate .

Isothermal titration calorimetry (ITC): This technique can characterize thermodynamic parameters of substrate binding. For example, E. coli QueF binds preQ0 in a strongly exothermic process (ΔH = -80.3 kJ/mol; -TΔS = 37.9 kJ/mol) with a Kd of 39 nM .

Substrate analogue studies: Chemical synthesis of substrate analogues such as 7-formyl-7-deazaguanine (a carbonyl analogue of the imine intermediate) allows probing of specific binding interactions. This analogue has been shown to bind weakly to queF (ΔH = -2.3 kJ/mol; -TΔS = -19.5 kJ/mol) but is not processed as a substrate .

QM/MM computational modeling: This approach has been successfully applied to model transition states, intermediates, and protein conformational changes along the reaction pathway, providing valuable insights for enzyme engineering and inhibitor design .

How does the catalytic efficiency of L. interrogans queF compare with queF from other bacterial species?

Comparative kinetic analysis of queF enzymes from different bacterial sources reveals important differences:

The relatively low kcat values of queF enzymes (0.011-0.12 s⁻¹) are comparable to other enzymes in the queuosine biosynthetic pathway, suggesting this may be a general feature of this pathway . The extremely low Km values for preQ0 (< 1 μM) indicate very high substrate affinity, which aligns with the Km of the subsequent enzyme TGT for preQ1 (0.39 μM) .

What unexpected enzymatic activities have been discovered for queF besides nitrile reduction?

In addition to its canonical nitrile reductase activity, queF has been found to possess a surprising "moonlighting" enzymatic activity:

NADPH hydratase activity: E. coli queF can catalyze the hydration of the C5=C6 double bond in the dihydronicotinamide moiety of NADPH, forming NADPHX. This enzymatic C6-hydrated NADPH exists as a 3.5:1 mixture of R and S forms and rearranges spontaneously through anomeric epimerization and cyclization. This activity appears to involve Cys190, the same catalytic nucleophile used for nitrile reduction, which acts as a general acid for protonation at the dihydronicotinamide C5 of NADPH .

This secondary activity is specific for NADPH; NADH and other 1,4-dihydronicotinamide derivatives are not accepted as substrates. This discovery reveals mechanistic similarities between queF's hydratase activity and natural C=C double bond hydration by dedicated hydratases .

What are the methodological challenges in working with recombinant L. interrogans queF?

Researchers working with recombinant L. interrogans queF should be aware of several technical challenges:

Storage stability: Like many recombinant proteins, queF stability can be compromised by repeated freeze-thaw cycles. Recommendations include storing working aliquots at 4°C for up to one week, with long-term storage at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months, while liquid preparations are stable for approximately 6 months when properly stored .

Activity assay limitations: When determining the Km for preQ0, researchers may encounter difficulties obtaining accurate rate data at concentrations below 1 μM due to poor signal-to-noise ratios at such low substrate concentrations. This challenge necessitates alternative approaches for accurate kinetic characterization .

Reconstitution protocols: For optimal activity, reconstitution of lyophilized queF should follow specific protocols, including centrifugation prior to opening the vial, reconstitution in deionized sterile water to 0.1-1.0 mg/mL, and addition of glycerol (5-50% final concentration) for stability .

Measurement of extremely low Km values: The exceptionally high affinity of queF for preQ0 (Km < 1 μM) presents methodological challenges for accurate determination of kinetic parameters and requires specialized approaches .

How might queF be engineered for broader substrate specificity or enhanced catalytic activity?

Engineering queF for expanded functionality requires understanding of several key aspects:

Critical residues for substrate recognition: The cysteine nucleophile (Cys190 in E. coli, Cys55 in B. subtilis) is essential for catalysis, but other residues involved in substrate recognition could be targets for mutagenesis to alter specificity .

Transition state stabilization: QM/MM calculations have identified residues involved in stabilizing transition states during catalysis. Mutations targeting these residues might enhance catalytic efficiency for the native reaction or enable new reactions .

Structural comparison with FolE family: Despite catalyzing different reactions, queF shares significant sequence homology with the type I GTP cyclohydrolases (FolE family). Understanding the structural differences responsible for their divergent substrate specificities and chemistries could inform rational design efforts .

Half-sites reactivity: E. coli queF exhibits half-of-the-sites reactivity in its homodimeric form. Engineering approaches might target full site utilization to potentially double catalytic efficiency .

While industrial applications are outside the scope of this FAQ, it's worth noting that the ability of queF to catalyze nitrile reduction under mild conditions makes it potentially valuable for biocatalytic applications, particularly if it can be engineered to accept a broader range of nitrile-containing substrates .

What controls should be included when characterizing recombinant L. interrogans queF activity?

Rigorous experimental design for queF characterization should include:

Negative controls:

• Reactions without enzyme to account for non-enzymatic preQ0 reduction or NADPH oxidation

• Heat-inactivated enzyme controls to confirm observed activity is enzymatic

• Substrate analogues that should not be processed (e.g., 7-formyl-7-deazaguanine)

Positive controls:

• Well-characterized queF from model organisms (E. coli or B. subtilis) tested under identical conditions

• Known queF inhibitors, if available, to confirm specificity of the assay

Validation approaches:

• Multiple independent methods to confirm activity (UV-based assay, HPLC analysis, NMR confirmation)

• Isotope labeling studies (e.g., deuterated NADPH) to confirm reaction mechanism

• Site-directed mutagenesis of the catalytic cysteine as a negative control

How can researchers distinguish between catalytic activity of queF and other NADPH-dependent enzymes in complex biological samples?

Differentiation strategies include:

Substrate specificity: QueF's high specificity for preQ0 can be leveraged. Few other enzymes recognize this substrate, making it a distinctive marker for queF activity.

Inhibition profiles: Chemical compounds that selectively inhibit queF but not other NADPH-dependent enzymes can help distinguish its activity.

Cysteine modification: Selective alkylation of the catalytic cysteine with iodoacetamide can specifically inactivate queF while leaving many other NADPH-dependent enzymes unaffected .

Immunoprecipitation: Using antibodies against queF to selectively remove it from complex samples before activity measurements.

Genetic approaches: In genetic systems where it's applicable, knockout or knockdown of queF can confirm the specificity of observed NADPH-dependent nitrile reductase activity.

What considerations are important when designing experiments to study the role of queF in Leptospira pathogenesis?

When investigating the potential role of queF in Leptospira pathogenesis, researchers should consider:

Growth conditions: L. interrogans growth is dependent on media supplemented with cobalamin, which affects various metabolic pathways . Consider how media composition might influence queF expression and activity.

Gene expression analysis: Compare queF expression levels under various conditions relevant to the infection cycle (temperature shifts, pH changes, osmolarity variations).

Genetic manipulation approaches: If developing knockout or knockdown strategies, consider the potential essentiality of queF. In many bacteria, disruption of tRNA modification pathways can significantly impair growth or stress responses.

Animal infection models: The hamster model has been successfully used to study L. interrogans pathogenesis and evaluate recombinant proteins as potential vaccines . Consider how queF function might be assessed in such models.

Immune response characterization: Determine if queF elicits antibody responses during infection and whether these antibodies have functional consequences for bacterial survival or virulence.

What are the latest approaches for investigating the role of queF-mediated tRNA modification in Leptospira biology?

State-of-the-art methods to study queF's role in tRNA modification include:

tRNA sequencing: Next-generation sequencing approaches specifically optimized for tRNA can identify the presence and abundance of queF-dependent modifications.

Mass spectrometry: High-resolution MS techniques can quantify modified nucleosides in tRNA populations under different conditions.

Ribosome profiling: This technique can reveal how queF-dependent tRNA modifications affect translation efficiency and accuracy, particularly at codons read by queuosine-modified tRNAs.

Comparative genomics: Analysis of queF conservation and genetic context across Leptospira species and serovars can provide insights into its evolutionary importance.

Metabolomic approaches: Untargeted metabolomics can identify downstream effects of queF activity on cellular metabolism, particularly pathways involving queF-modified tRNAs.