THE PHOSPHOGLYCERATE KINASE AND GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE GENES FROM THE THERMOPHILIC ARCHAEON SULFOLOBUS-SOLFATARICUS OVERLAP BY 8-BP - ISOLATION, SEQUENCING OF THE GENES AND EXPRESSION IN ESCHERICHIA-COLI

The overlapping genes encoding phosphoglycerate kinase (PGK) and glyce raldehyde-3-phosphate dehydrogenase (GraP-DH) from the hyperthermophil ic archaeon Sulfolobus solfataricus have been cloned and sequenced. PC R primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the pgk gene. This was the n used to screen a genomic library to isolate the full length pgk gene . A 2.5-kb BglII fragment of S. solfataricus DNA contained both the pg k gene and the gap gene immediately downstream. Unexpectedly, the pgk and gag genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene. Evid ence that the two genes are co-transcribed was obtained by Northern-bl ot analysis. The S. solfataricus PGK amino acid sequence shows 43% and 45% identity to the PGK sequences of the Archaea Methanobacterium bry antii and Methanothermus fervidus, respectively. High level expression of the S. solfataricus PGK and GraP-DH in Escherichia cell was achiev ed, with heat treatment at 80 degrees C proving an effective first ste p in the purification of these recombinant enzymes from extracts of th e E. coli host. Purified recombinant S. solfataricus PGK and GraP-DH s howed half lives of 39 min and 17 h, respectively, at 80 degrees C. Un like bacterial GraP-DH enzymes, S. solfataricus GraP-DH was able to us e both NAD(+) and NADP(+) as cofactors, but exhibited a marked prefere nce for NADP(+).