Recombinant Thermococcus gammatolerans Diphthine synthase (dphB)

What is diphthine synthase and what is its role in diphthamide biosynthesis?

Diphthine synthase (often labeled as Dph5 in literature) catalyzes the second step in diphthamide biosynthesis. Diphthamide is a unique post-translational modification found on histidine residues in eukaryotic and archaeal translation elongation factor 2 (EF2). The biosynthesis pathway involves three distinct steps:

• First step: Transfer of 3-amino-3-carboxypropyl group from S-adenosyl-L-methionine (SAM) to the target histidine residue

• Second step: Trimethylation of the amino group by diphthine synthase to form diphthine

• Third step: ATP-dependent amidation of the carboxyl group

Diphthine synthase specifically catalyzes the methylation reactions required to convert the 3-amino-3-carboxypropyl-modified histidine to diphthine by transferring methyl groups from SAM in a sequential manner .

How does Thermococcus gammatolerans diphthine synthase compare structurally to other archaeal diphthine synthases?

While detailed structural data specific to T. gammatolerans diphthine synthase is limited, we can draw insights from studies on related archaeal species. Archaeal diphthine synthases are typically smaller than their eukaryotic counterparts, with molecular weights around 30 kDa, as demonstrated with P. horikoshii Dph5 .

The archaeal enzyme likely shares the following characteristics:

• SAM-binding domain with conserved motifs

• Recognition sites for interacting with modified EF2

• Ability to perform processive methylation (mono-, di-, and tri-methylation)

As T. gammatolerans is a hyperthermophilic and radioresistant archaeon, its diphthine synthase would be expected to demonstrate enhanced thermostability compared to mesophilic counterparts .

What substrate specificity does T. gammatolerans diphthine synthase exhibit?

T. gammatolerans diphthine synthase would be expected to demonstrate high substrate specificity similar to P. horikoshii Dph5, which specifically recognizes EF2 with the 3-amino-3-carboxypropyl modification on the target histidine residue. Studies with P. horikoshii have shown:

• The enzyme does not act on unmodified EF2

• It requires the product of the first step of diphthamide biosynthesis

• It recognizes specific regions surrounding the modified histidine

The substrate specificity is critical for experimental design when assaying enzyme activity, as the substrate needs to be appropriately pre-modified by the first step enzyme (Dph2) .

What expression systems are most effective for recombinant T. gammatolerans diphthine synthase?

Based on successful approaches with related archaeal proteins:

Recommended expression system:

• Vector: pET-28a(+) with N-terminal His-tag for purification

• Host: E. coli BL21(DE3) or Rosetta(DE3) for rare codon optimization

• Induction: 0.5 mM IPTG at OD600 of 0.6-0.8

• Expression temperature: 18-25°C for 16-20 hours to enhance solubility

Optimization considerations:

• Codon optimization may be necessary due to differences between archaeal and E. coli codon usage

• Addition of chaperones (GroEL/GroES) may improve folding of the thermostable protein

• For cloning, primers should target the full coding sequence of dphB, similar to the approach used for P. horikoshii Dph5

What purification protocol yields optimal purity and activity for recombinant T. gammatolerans diphthine synthase?

A multi-step purification strategy is recommended:

• Immobilized metal affinity chromatography (IMAC):

  • Buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol

  • Wash with 20-40 mM imidazole

  • Elute with 250-300 mM imidazole gradient

• Ion exchange chromatography:

  • Q-Sepharose column for anion exchange

  • Buffer: 20 mM Tris-HCl (pH 8.0), 50-500 mM NaCl gradient

• Size exclusion chromatography:

  • Superdex 200 column

  • Buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5% glycerol

Purification should be monitored by SDS-PAGE, with expected molecular weight around 30 kDa based on P. horikoshii Dph5 .

How should recombinant T. gammatolerans diphthine synthase be stored to maintain activity?

Given the thermostable nature of proteins from T. gammatolerans:

Short-term storage (1-2 weeks):

• 4°C in storage buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM DTT, 10% glycerol)

Long-term storage:

• Flash freeze aliquots in liquid nitrogen

• Store at -80°C with 20% glycerol as cryoprotectant

• Avoid repeated freeze-thaw cycles

Activity retention testing:

• Periodically test enzyme activity using SAH formation assay

• Monitor protein aggregation by dynamic light scattering

What are the optimal reaction conditions for in vitro activity of T. gammatolerans diphthine synthase?

Based on studies with P. horikoshii Dph5, the following conditions are recommended:

Reaction buffer:

• 100 mM Tris-HCl (pH 8.0)

• 75 mM NaCl

• 50 mM KCl

• 1 mM EDTA

• 5 mM DTT

• 5 mM MgCl₂

Temperature optimization:

• Test activity range between 37-80°C

• P. horikoshii Dph5 showed activity at 37°C, but T. gammatolerans enzymes may function optimally at higher temperatures given its growth temperature optimum of 88°C

Substrate concentrations:

• Modified EF2: 30 μM

• SAM: 50-100 μM

• Enzyme: 30-60 μM

How can methyltransferase activity of T. gammatolerans diphthine synthase be detected and measured?

Multiple complementary approaches can be used:

1. SAH formation detection by HPLC:

• Run reaction with SAM and substrate

• Stop reaction with 5% TFA

• Remove precipitated proteins by centrifugation

• Analyze supernatant by HPLC on a C18 column

• Monitor absorbance at 260 nm

• Use linear gradient from 0 to 40% buffer B in 20 min (Buffer A: 50 mM ammonium acetate, pH 5.4; Buffer B: 50% v/v methanol/water)

• SAH standard should elute around 10 minutes

2. Radioactive assay using methyl-¹⁴C-SAM:

• Incubate enzyme with substrate and methyl-¹⁴C-SAM

• Resolve reaction by SDS-PAGE without heat denaturation

• Detect incorporation by autoradiography

• Note: This approach may not be effective if elimination of the trimethylamino group occurs rapidly

What analytical techniques can confirm successful modification by T. gammatolerans diphthine synthase?

Mass spectrometry approaches:

• MALDI-MS analysis:

  • Digest modified protein with trypsin

  • Analyze peptide fragments

  • Look for mass shifts corresponding to:

    • Mono-methylation: +14 Da

    • Di-methylation: +28 Da

    • Tri-methylation: +42 Da

  • Be aware that trimethylated product may undergo elimination, resulting in a different mass signature

• Tandem MS/MS:

  • For detailed structural characterization

  • Can confirm specific modification sites

  • Useful for distinguishing between various possible products

Expected masses for T. gammatolerans modified peptides:
The exact masses would depend on the specific peptide sequence containing the target histidine in T. gammatolerans EF2, but the principle is similar to P. horikoshii, where:

• Unmodified peptide: 1545.80 m/z

• ACP-modified peptide: 1646.84 m/z

• After elimination of trimethylamino group: 1629.77 m/z

How does the stability of T. gammatolerans diphthine synthase compare to other archaeal homologs?

T. gammatolerans is known for its extreme radioresistance and thermophilic nature, suggesting its diphthine synthase would have enhanced stability properties:

Expected stability features:

• Higher thermal stability than P. horikoshii Dph5

• Potential resistance to oxidative damage

• Likely maintains activity at temperatures approaching 80-90°C

• May exhibit stability in the presence of denaturants and organic solvents

Comparative stability assessment methodology:

• Thermal shift assays using differential scanning fluorimetry

• Activity retention after exposure to different temperatures

• Half-life determination at various temperatures

• Resistance to chemical denaturants

What strategies can address the elimination of the trimethylamino group from the diphthine product?

The elimination of the trimethylamino group from diphthine presents a significant challenge in studying the complete diphthamide pathway. Based on observations with P. horikoshii Dph5, the following strategies may be effective:

Preventing elimination:

• Lower reaction temperature (though this may reduce enzyme activity)

• Modify buffer conditions to stabilize the trimethylated product

• Explore rapid coupling with the third enzymatic step to prevent accumulation of diphthine

• Consider enzyme engineering to modify the active site environment

Analyzing elimination products:

• The elimination likely proceeds through a mechanism similar to Hofmann or Cope elimination

• The resulting product contains a 3-carboxy-2-propenyl group on the histidine residue

• This modification can be detected by a mass shift from 1688.87 to 1629.77 m/z (based on P. horikoshii data)

How can the complete diphthamide biosynthesis pathway be reconstituted using T. gammatolerans enzymes?

Reconstituting the complete pathway requires careful coordination of all three enzymatic steps:

Step 1: ACP transfer (Dph2-catalyzed reaction)

• Express and purify recombinant T. gammatolerans Dph2

• Reconstitute [4Fe-4S] cluster under anaerobic conditions

• Incubate with EF2 substrate and SAM at optimal temperature

• Confirm modification by mass spectrometry

Step 2: Trimethylation (Dph5/dphB-catalyzed reaction)

• Buffer exchange to optimal conditions for diphthine synthase

• Add purified T. gammatolerans diphthine synthase and SAM

• Monitor reaction by SAH formation

Step 3: Amidation

• Add ATP and amidation enzyme (if identified in T. gammatolerans)

• Alternatively, use ATP, creatine phosphate, and phosphocreatine kinase

Key considerations for complete pathway:

• Temperature compatibility between different enzymes

• Buffer conditions suitable for all enzymatic steps

• Rapid progression between steps to prevent loss of intermediates

• Analysis of final product by mass spectrometry and functional assays