Recombinant Acinetobacter sp. Thiazole synthase (thiG)

Basic Research Questions

• What is the role of thiazole synthase (thiG) in Acinetobacter species metabolism?

  Thiazole synthase (thiG) in Acinetobacter species catalyzes a critical step in thiamine (vitamin B1) biosynthesis. Specifically, thiG catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine . The enzyme forms a tetramer with 222 symmetry, and the monomer adopts a (betaalpha)8 barrel structure showing similarities to aldolase class 1 and flavin mononucleotide-dependent oxidoreductase families . In Acinetobacter baumannii, thiG is 261 amino acids long with a molecular weight of approximately 27.8 kDa . Thiamine is essential for carbohydrate metabolism, and its deficiency significantly impacts bacterial survival and virulence.

• How can recombinant thiG from Acinetobacter be expressed and purified for research purposes?

  Expression and purification of recombinant thiG from Acinetobacter species can be achieved using several heterologous expression systems:

  Expression System	Advantages	Considerations	Tag Options
  E. coli	High yield, rapid growth	Potential inclusion body formation	His-tag, Avi-tag
  Yeast	Post-translational modifications	Longer cultivation time	Various tags available
  Baculovirus	Better folding for complex proteins	More expensive	Multiple tag options
  Mammalian cells	Closest to native conditions	Most expensive, lower yield	Various tag options

  The thiG gene (typically 786 bp encoding the 261 amino acid protein) can be amplified from Acinetobacter genomic DNA using PCR with specific primers. For bacterial expression, the gene is typically cloned into vectors like pET or pBAD series. Recombinant thiG can be purified using affinity chromatography (if tagged), followed by ion-exchange and size-exclusion chromatography to achieve high purity .

• What are the standard assays to measure thiG enzymatic activity?

  Several methodological approaches can be employed to measure thiG activity:

  • Spectrophotometric assays: Monitoring the consumption of DXP or formation of the thiazole product at specific wavelengths.

  • High-Performance Liquid Chromatography (HPLC): Quantifying reaction products after separation.

  • Mass spectrometry: Detecting the formation of thiazole phosphate with high sensitivity.

  • Coupled enzyme assays: Using auxiliary enzymes to couple thiG activity to a measurable event (e.g., NADH oxidation).

  • Thermodynamic binding assays: Using techniques like Surface Plasmon Resonance (SPR) to measure substrate binding, as performed with inhibitor studies where Z24 was shown to bind to thiazole synthase in a dose-dependent manner .

• What are the requirements for NIH biosafety guidelines when working with recombinant Acinetobacter thiG?

  Research involving recombinant Acinetobacter thiG must comply with NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules:

  • Experiments involving recombinant Acinetobacter require IBC (Institutional Biosafety Committee) approval before initiation .

  • Acinetobacter baumannii is considered an opportunistic pathogen with multidrug resistance potential, requiring appropriate biosafety measures .

  • Work with recombinant thiG should be conducted at Biosafety Level 2 (BSL-2) containment at minimum.

  • All personnel must receive proper training in handling recombinant DNA and potentially infectious materials.

  • Proper waste disposal procedures must be followed according to institutional and NIH guidelines.

  • If your research is NIH-funded, compliance with these guidelines is a condition of funding .

• How does thiG structure differ between Acinetobacter species and other bacteria?

  Thiazole synthase from Acinetobacter baumannii shares the conserved ThiG family structure but with some notable differences:

  • The full length is 261 amino acids with a predicted molecular weight of 27.8 kDa .

  • The protein adopts a (betaalpha)8 barrel structure similar to other bacterial thiG proteins .

  • Key active site residues (equivalent to Glu98 and Asp182 identified in other bacterial thiG proteins) are conserved and essential for thiazole formation .

  • ThiG functions in complex with the sulfur carrier protein ThiS, which has a fold similar to ubiquitin .

  • Acinetobacter thiG shares high sequence homology with other proteobacterial thiG proteins but has specific variations in surface-exposed residues that could be exploited for species-specific inhibitor design.