Sequence and expression of the gene encoding 3-phosphoglycerate kinase from Bacillus stearothermophilus

Abstract

The structural gene (pgk) encoding 3-phosphoglycerate (PGK) from Bacillus stearothermophilus NCA1503, has been cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 394 amino acids (aa) (calculated Mr 42,703) and, in common with other prokaryotic pgk genes, is preceded by the structural gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Constructs containing the B. stearothermophilus pgk gene and its flanking sequences in the high-copy plasmid, pUC9, co-express both PGK and GAPDH at high levels in transformed E. coli cells, typically producing PGK at levels of up to 30% of the soluble cell protein. The deduced aa sequence of B. stearothermophilus PGK is compared with those of the mesophilic (yeast) and extreme thermophilic (Thermus thermophilus) enzymes since the crystal structure of these PGKs are known or are in the process of being determined. Changes in the sequences of the three enzymes, as they appear to relate to the enhancement of thermal stability, are discussed.