Recombinant Deoxyguanosinetriphosphate triphosphohydrolase-like protein (dgt)

What is Deoxyguanosinetriphosphate triphosphohydrolase (Dgt) and what is its primary catalytic activity?

Dgt is an enzyme encoded by the dgt gene in Escherichia coli that catalyzes the hydrolysis of dGTP (2′-deoxyguanosine-5′-triphosphate) into deoxyguanosine (dG) and triphosphate (PPPi) . While the enzyme shows high specificity for dGTP, it can also hydrolyze other canonical and noncanonical dNTPs with lower efficiency . The enzyme plays a critical role in regulating cellular dNTP pools, particularly dGTP, which directly impacts DNA polymerase fidelity and cellular mutation rates .

What is the quaternary structure of E. coli Dgt and how does it compare to other similar enzymes?

E. coli Dgt forms a hexameric structure as determined by crystallographic studies at 3.1 Å resolution . This hexameric assembly represents a significant structural difference from its human homolog SAMHD1, which forms a tetramer . The Dgt hexamer contains a central pore and DNA-binding regions that play important roles in its regulation . The hexameric structure permits cooperative interactions between subunits, which is reflected in the sigmoidal kinetics observed in the absence of DNA .

How does deletion of the dgt gene affect bacterial cells?

Deletion of the dgt gene has been shown to:

• Increase cellular dGTP pool by approximately 2-fold

• Generate a mutator phenotype with increased frequencies of specific base pair substitution mutations

• Impact the thymine-salvage pathway due to altered deoxyguanosine production

These findings indicate that while not essential for viability under standard conditions, Dgt plays a significant role in maintaining genomic integrity by regulating nucleotide pools .

How does DNA binding affect Dgt enzymatic activity?

DNA binding significantly alters Dgt's enzymatic properties through several mechanisms:

• Kinetic profile change: In the absence of DNA, Dgt displays sigmoidal (cooperative) kinetics with an apparent Km of 33 ± 1.5 μM for dGTP. Upon DNA binding, the enzyme shows hyperbolic (Michaelis-Menten) kinetics with a reduced Km of 10 ± 1.5 μM .

• Conformational changes: DNA binding induces significant structural changes in the enzyme, including alterations in the catalytic site. Specifically:

  • Helices α13 (closest to DNA) and α10 (adjacent to the active site) undergo conformational shifts

  • Helix α10 straightens, moving Tyr-272 approximately 2.4 Å away from the dGTP substrate, potentially facilitating substrate binding

  • Arg-442' from an adjacent monomer moves closer to the dGTP substrate

These DNA-induced conformational changes explain how DNA binding enhances Dgt activity, particularly at low substrate concentrations .

What is the DNA binding specificity of Dgt?

Dgt demonstrates high affinity and specificity for single-stranded DNA (ssDNA) but shows no affinity for RNA molecules. Fluorescence polarization experiments have established:

• Tight binding to 22-mer ssDNA with a Kd of 52 nM

• No measurable binding to ssRNA of the same length and sequence

• Preference for longer DNA strands (e.g., bacteriophage M13), suggesting Dgt can form chains of hexamer molecules along DNA strands

This DNA-binding specificity may be physiologically relevant to situations where ssDNA becomes exposed, such as during replication fork stalling or DNA damage .

How does GTP inhibit Dgt activity?

GTP acts as a competitive inhibitor of Dgt, with the following characteristics:

• It increases the dGTP Km while leaving kcat unaffected

• The GTP-binding constant derived from kinetic studies is 120 ± 50 μM

• This binding affinity indicates Dgt will readily bind GTP in vivo, where GTP is present at high micromolar to low millimolar concentrations

• GTP has been shown to bind directly to the Dgt active site, competing with dGTP

• This competition means the effective Km for dGTP in vivo would be significantly higher than the in vitro value (≤20 μM) measured in the absence of GTP

The competitive inhibition by GTP may represent a physiological regulatory mechanism for controlling Dgt activity in cells .

What is the mechanism by which T7 bacteriophage protein Gp1.2 inhibits Dgt?

Gp1.2 is a T7 bacteriophage protein that inhibits Dgt through a complex mechanism:

• Inhibition parameters:

  • Gp1.2 alone shows an IC50 of 160 ± 20 nM under kcat,app conditions

  • When 1 mM GTP is included, the Gp1.2 IC50 decreases significantly to 27 ± 3 nM

  • This indicates a synergistic inhibitory effect between GTP and Gp1.2

• Mechanistic aspects:

  • Gp1.2 can bind to both free and substrate-bound Dgt, suggesting a mixed-type inhibition

  • Gp1.2 fails to fully inhibit Dgt that is already bound to nucleic acids

  • The Dgt-Gp1.2 complex can still bind dGTP or GTP, but hydrolysis is prevented

This inhibition mechanism represents a viral strategy to counteract the host cell's defense against infection, as Dgt can restrict viral replication by limiting available dNTP pools .

What are the optimal protocols for purifying recombinant Dgt with minimal DNA contamination?

Based on the research literature, two protocols have been developed for Dgt purification with different DNA contamination profiles:

Protocol 1 (Standard protocol):

• Uses Bugbuster, lysozyme, and Benzonase for cell lysis

• May result in DNA contamination in the final preparation

Protocol 2 (DNA-free preparation):

• Uses Bugbuster, lysozyme, and 2M NaCl for lysis

• Omits Benzonase

• Includes PEG/dextran precipitation step (6% PEG 6000, 5% dextran 500) to remove DNA

• Uses Ni-NTA chromatography at room temperature

• Results in essentially DNA-free enzyme preparation

For structural studies or experiments where DNA might interfere, Protocol 2 is recommended to ensure the enzyme is free from bound DNA .

How can Dgt activity be measured in vitro?

Dgt activity can be measured using an enzyme-coupled spectrophotometric assay:

Reaction components:

• 100 mM Tris-HCl, pH 8.0

• 5 mM MgCl2

• 50 milliunits/ml purine nucleoside phosphorylase

• 500 milliunits/ml xanthine oxidase

• 5 mM sodium phosphate

• Variable dGTP concentrations

• 4 nM Dgt (final concentration)

Reaction principle:

• Dgt hydrolyzes dGTP to deoxyguanosine

• Purine nucleoside phosphorylase and xanthine oxidase convert deoxyguanosine to 8-oxoguanine

• Formation of 8-oxoguanine is monitored continuously at 297 nm

• Reaction rate is determined from the linear slope of absorbance increase

For studying the effect of DNA on activity, a single-stranded DNA oligonucleotide (e.g., 40-mer) can be included in the reaction mixture, with pre-incubation of the enzyme with DNA for 20 minutes at 37°C before initiating the reaction .

How can DNA-binding properties of Dgt be analyzed experimentally?

DNA binding can be analyzed using fluorescence polarization:

Method overview:

• Use DNA-free Dgt preparation

• Prepare fluorescently labeled ssDNA (typically 22-mer)

• Titrate increasing amounts of Dgt into a solution containing labeled DNA

• Measure changes in fluorescence anisotropy, which increases upon protein binding due to decreased rotational mobility of the DNA

• Fit data to a binding curve using a quadratic equation to calculate Kd values

This approach has demonstrated that wild-type Dgt binds 22-mer ssDNA with a Kd of 52 nM, indicating a tight protein-DNA complex, while showing no measurable binding to RNA of the same sequence .

How can DNA binding-defective mutants of Dgt be generated and what are their properties?

DNA binding-defective mutants can be created by site-directed mutagenesis targeting residues in the DNA-binding cleft:

Mutation strategy:

• In one studied case, S34D and G37E double substitutions were introduced in the DNA-binding cleft

• These mutations introduce significant electrostatic repulsion toward the DNA backbone

Properties of the S34D/G37E mutant:

• Quaternary structure: Retains hexameric form similar to wild-type protein

• DNA binding: No binding to fluorescently labeled ssDNA in fluorescence anisotropy assays

• Enzymatic activity:

  • Active with increased catalytic efficiency at low substrate concentrations

  • Apparent Km of 16 μM (compared to 33 μM for wild-type)

  • No stimulation by added DNA

In vivo effects:

• The mutant displays a modest mutator effect in a mismatch-repair-defective background

• Expression of the mutant protein from a pET vector is toxic to cells, even under modest overexpression conditions

These findings highlight the physiological importance of DNA binding for Dgt function in vivo .

What is the relationship between Dgt, dNTP pool regulation, and mutagenesis?

Dgt plays a critical role in controlling cellular dNTP pools and mutation rates:

• dGTP pool regulation:

  • Deletion of dgt leads to ~2-fold increase in cellular dGTP levels

  • Overexpression can reduce dGTP levels by ~5-fold

• Mutation effects:

  • Δdgt strains exhibit a mutator phenotype with increased frequencies of specific base-pair substitutions

  • This phenotype is consistent with observations that imbalanced dNTP pools compromise DNA polymerase fidelity

• Mechanism of fidelity maintenance:

  • By selectively hydrolyzing dGTP, Dgt helps maintain balanced dNTP pools

  • Balanced pools are essential for accurate DNA replication

  • Imbalanced pools can alter the kinetics of nucleotide incorporation during replication, leading to increased error rates

The connection between dNTP pool maintenance and mutation rates highlights the critical role of Dgt in genomic integrity .

How does Dgt function in bacterial defense against viral infection?

Dgt may serve as a host defense mechanism against viral infections:

• Anti-viral mechanism:

  • By restricting dNTP pools, Dgt can limit the resources available for viral genome replication

  • Viruses replicate their genomes using the host cell's dNTP pool

  • dNTPases like Dgt can function as restriction factors by hydrolyzing dNTPs needed for viral replication

• Viral counter-strategies:

  • Some viruses have evolved specific inhibitors to counteract host dNTPases

  • T7 bacteriophage produces protein Gp1.2, which specifically inhibits Dgt

  • This inhibition prevents the host from restricting viral replication through dNTP depletion

This host-virus interaction parallels other systems, such as human SAMHD1 (a dNTPase) which restricts HIV replication, and HIV Vpx protein which counteracts SAMHD1 .

What are the unresolved questions regarding Dgt function and regulation?

Several important aspects of Dgt biology remain to be fully elucidated:

• In vivo DNA binding:

  • How much Dgt in actively growing cells is actually DNA-bound?

  • How does Dgt access single-stranded DNA in vivo when competing with single-stranded DNA-binding proteins?

  • Under what conditions might Dgt gain access to genomic DNA (e.g., during DNA damage or replication stress)?

• Regulatory mechanisms:

  • How is Dgt activity regulated in response to cellular needs?

  • What is the rate-limiting step for Dgt activity in cells?

  • Are there additional cellular factors that modulate Dgt activity?

• Physiological roles:

  • What is the broader role of dNTP triphosphohydrolases in bacterial metabolism?

  • How does Dgt activity integrate with other aspects of nucleotide metabolism?

  • Are there additional biological functions beyond dGTP pool maintenance?

Further research in these areas will provide a more complete understanding of Dgt function and its importance in bacterial physiology .

How can structural information about Dgt be used to design specific inhibitors or activators?

The available structural data on Dgt opens possibilities for rational design of modulators:

• Key structural features for targeting:

  • The active site containing the dGTP binding pocket

  • The DNA binding cleft

  • The hexamer interfaces important for cooperative activity

  • Allosteric sites involved in DNA-mediated activation

• Potential approaches:

  • Design of competitive inhibitors that mimic dGTP or GTP

  • Development of compounds that prevent DNA binding and subsequent activation

  • Creation of molecules that disrupt the hexameric structure

  • Exploration of the Arg-442' residue from adjacent monomers as a target for inhibitor design

• Applications:

  • Inhibitors might be useful as antimicrobial agents that induce mutagenesis

  • Activators could potentially enhance dGTP depletion and limit viral replication

  • Modulators could serve as tools to study the physiological consequences of altered Dgt activity

The unique structural features of Dgt, particularly its DNA-binding properties and hexameric organization, provide opportunities for developing specific modulators with potential research and therapeutic applications .