Pyruvate phosphate dikinase from a thermophilic actinomyces Microbispora rosea subsp. aerata: purification, characterization and molecular cloning of the gene

Abstract

Various thermophilic bacteria were analyzed by Southern hybridization analysis using oligonucleotide probes coding for the pyruvate phosphate dikinase (PPDK) gene from Clostridium symbiosum, and positive hybridization signals were observed in the chromosomal DNAs from Microbispora rosea subsp. aerata (IFO 14047). PPDK activity was detected in lactose induced cells and the enzyme was purified to homogeneity. The molecular mass of PPDK was estimated to be 230 000 by gel filtration chromatography and 91 000 by SDS–PAGE, suggesting that PPDK is a dimeric enzyme. This enzyme was specific for adenine nucleotide and the apparent K m values for AMP, PP i, and phosphoenolpyruvate were 5, 38, and 280 μM, respectively. It was stable in the pH range 6 to 11, and retained 80% activity after 60 min heat treatment at 60°C. We cloned the PPDK gene from M. rosea. It consists of 878 amino acids with a molecular mass of 95 514. Sequence comparison indicates around 50% similarity with other PPDKs and it has all the highly conserved regions of the related enzymes. We expressed the PPDK gene in Escherichia coli and obtained enzymatically active protein.