Summary auto-generated
Researchers isolated and purified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Clostridium acetobutylicum, a bacterium used industrially for butanol production. The enzyme was purified 56-fold using ammonium sulfate precipitation, gel filtration, and chromatography techniques, achieving a specific activity of 27 U/mg. Biochemical characterization revealed the enzyme has a tetrameric structure (native mass 160 kDa, subunit mass 40 kDa) with optimal activity at pH 8.5-9.3 and temperature of 50°C. The researchers cloned and sequenced the gap gene encoding GAPDH and discovered it clustered with three other complete glycolytic genes (pgk, tpi, pgm(i)) and a partial upstream open reading frame, all transcribed in the same direction. The absence of transcription terminator sequences between genes suggests they form a single operon. Complementation experiments in an Escherichia coli gap mutant confirmed the clostridial gene's functionality. The clostridial GAPDH shows high sequence conservation with GAPDHs from other organisms, particularly 95% identity with C. pasteurianum, and maintains critical residues for catalytic function and cofactor binding.
Key findings
- GAPDH from C. acetobutylicum is a homotetrameric enzyme (160 kDa native) composed of 40 kDa subunits with specific activity of 27 U/mg and pH optimum between 8.5-9.3
- The gap gene clusters with three other glycolytic genes (pgk, tpi, pgm(i)) in the same transcriptional orientation with no typical transcription terminators, suggesting a common operon
- The clostridial GAPDH is moderately thermotolerant (50°C optimum) and shows high sequence identity to homologous enzymes from other organisms with conserved catalytic and structural residues
- The gap gene successfully complemented an E. coli gap mutant, confirming its functionality in a heterologous host
- GAPDH expression shows minimal regulation between acidogenic and solventogenic growth phases, suggesting post-transcriptional control mechanisms
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Abstract
Glyceraldehyde-3-phosphate dehydrogenase was purified from Clostridium acetobutylicum by sequential ammonium sulfate precipitation, gel filtration and anion-exchange chromatography (to a specific activity of 27 U mg(-1)). The enzyme had a molecular mass of 40 kDa as determined by SDS-PAGE and a native molecular mass of 160 kDa as determined by nondenaturing PAGE, indicating that it has a homotetrameric composition. Its pH optimum was between 8.5 and 9.3. The corresponding gene (gap) was cloned and sequenced from C. acetobutylicum DSM 792 and found to cluster with other genes of enzymes from the glycolytic pathway (pgk, phosphoglycerate kinase; tpi, triosephosphate isomerase; pgm(i), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase). No sequences resembling rho-independent transcription terminators were found in the intergenic regions. A plasmid carrying the clostridial gap gene complemented an Escherichia coli gap mutant.