Recombinant Photorhabdus luminescens subsp. laumondii 6,7-dimethyl-8-ribityllumazine synthase (ribH)

Basic Research Questions

• What is the biochemical function of 6,7-dimethyl-8-ribityllumazine synthase (ribH) in Photorhabdus luminescens?

  6,7-dimethyl-8-ribityllumazine synthase (ribH) catalyzes the penultimate step in riboflavin (vitamin B2) biosynthesis. This enzyme specifically mediates the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione to form 6,7-dimethyl-8-ribityllumazine . The reaction represents a critical step in the metabolic pathway that ultimately produces riboflavin, an essential vitamin that serves as a precursor for flavin coenzymes (FAD and FMN) involved in numerous redox reactions central to P. luminescens metabolism.

  The methodological approach to confirm this function includes:

  1. Gene cloning and recombinant expression in E. coli

  2. Protein purification via affinity chromatography

  3. In vitro enzyme activity assays measuring substrate consumption and product formation via HPLC

  4. Complementation studies in ribH-deficient bacterial strains to confirm functional activity

• How is the ribH gene organized in the P. luminescens genome and what is its regulatory context?

  Based on comparative analysis with other bacterial species, the ribH gene in P. luminescens is likely located within a riboflavin biosynthesis operon. In P. luminescens TT01, the complete genome sequence (5,688,987 base pairs) contains 4,839 predicted protein-coding genes . Similar to what has been observed in Brucella species, the ribH gene may be located inside a small riboflavin operon, together with other riboflavin biosynthesis genes and possibly the nusB gene (which encodes an antitermination factor) .

  The methodological approach to characterize the genomic organization includes:

  1. Bioinformatic analysis using tools like BLAST and SMART to identify the ribH gene and surrounding regions

  2. Promoter prediction analysis to identify potential regulatory elements

  3. RT-PCR to determine if ribH is co-transcribed with neighboring genes

  4. Reporter gene assays (such as using the pPINT-mCherry system) to analyze promoter activity

• What expression systems are most effective for producing recombinant P. luminescens ribH protein?

  For optimal expression of recombinant P. luminescens ribH, several expression systems can be employed:

  Expression System	Vector	Induction	Temperature	Advantages
  E. coli BL21(DE3)	pET series	IPTG (0.1-1.0 mM)	16-30°C	High yield, well-established
  E. coli Arctic Express	pET or pTrc	IPTG (0.1 mM)	10-12°C	Improved folding for difficult proteins
  E. coli Rosetta	pET series	IPTG (0.5 mM)	18-25°C	Supplies rare codons

  Methodology for optimizing expression:

  1. Clone the ribH gene with an N- or C-terminal His-tag for purification

  2. Test multiple expression conditions (temperature, IPTG concentration, induction time)

  3. Analyze soluble versus insoluble fractions by SDS-PAGE

  4. Scale up production using the optimized conditions

  5. For difficult-to-express proteins, consider fusion partners like MBP or SUMO

• What purification strategies yield the highest purity and activity for recombinant ribH?

  A step-wise purification strategy would include:

  1. Cell lysis: Sonication or French press in buffer (typically 50 mM Tris-HCl pH 7.5-8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT)

  2. Clarification: Centrifugation at 15,000-20,000 × g for 30-45 minutes

  3. Affinity chromatography: Ni-NTA for His-tagged protein, with step gradient elution

  4. Size-exclusion chromatography: To separate oligomeric forms and remove aggregates

  5. Ion-exchange chromatography: For further purification if needed

  The final preparation should be analyzed by:

  • SDS-PAGE for purity assessment (typically >95%)

  • Enzymatic activity assays

  • Mass spectrometry for identity confirmation

  • Dynamic light scattering for homogeneity analysis

• How can the enzymatic activity of ribH be measured in vitro?

  Several complementary methods can be used to measure ribH activity:

  1. Spectrophotometric assay: Monitor formation of 6,7-dimethyl-8-ribityllumazine by absorbance increase at 408-410 nm

  2. HPLC analysis: Quantitatively measure substrate consumption and product formation

  3. Coupled enzymatic assay: Link lumazine synthase activity to riboflavin synthase activity and measure riboflavin fluorescence (excitation: 470 nm, emission: 530 nm)

  Standard reaction conditions:

  • Buffer: 100 mM potassium phosphate, pH 7.0-7.5

  • Temperature: 25-37°C (optimize for the specific enzyme)

  • Substrates: 3,4-dihydroxy-2-butanone 4-phosphate and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione

  • Enzyme concentration: 0.1-10 μM (optimize based on activity)

Advanced Research Questions