APBA, THE KETOPANTOATE REDUCTASE ENZYME OF SALMONELLA-TYPHIMURIUM IS REQUIRED FOR THE SYNTHESIS OF THIAMINE VIA THE ALTERNATIVE PYRIMIDINE BIOSYNTHETIC-PATHWAY

The apbA gene of Salmonella typhimurium was shown to encode ketopantoi c acid reductase. ApbA was purified from crude cell-free extracts to g reater than 95% homogeneity after two chromatographic steps. N-termina l amino acid sequencing (first 15 amino acids) and Western blot analys is confirmed the isolated protein was ApbA. The functional protein was a monomer with a molecular mass of 31.1 kDa. Optimal reaction conditi ons for the reduction of ketopantoic acid were established at a pH of 6.25, and a temperature of 42 degrees C. The preferred electron source was NADPH, and the apparent K-m constants of the enzyme for NADPH and ketopantoic acid were determined to be 0.776 +/- 0.09 mM and 0.742 +/ - 0.01 mM, respectively. The homogeneous enzyme had a specific activit y of 64.3.