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

Various thermophilic bacteria were analyzed by Southern hybridization analy sis 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. aerat a (IFO 14047). PPDK activity was detected in lactose induced cells and the enzyme was purified to homogeneity. The molecular mass of PPDK was estimate d to be 230 000 by gel filtration chromatography and 91 000 by SDS-PAGE, su ggesting that PPDK is a dimeric enzyme. This enzyme was specific for adenin e nucleotide and the apparent K-m values for AMP, PPi, and phosphoenolpyruv ate were 5, 38, and 280 mu M, respectively. It was stable in the pH range 6 to 11, and retained 80% activity after 60 min heat treatment at 60 degrees C. We cloned the PPDK gene from M. rosea. It consists of 878 amino acids w ith a molecular mass of 95 514. Sequence comparison indicates around 50% si milarity with other PPDKs and it has all the highly conserved regions of th e related enzymes. We expressed the PPDK gene in Escherichia coli and obtai ned enzymatically active protein. (C) 1999 Elsevier Science B.V. All rights reserved.