Phosphoglycerate mutases from microorganisms
Phosphoglycerate mutase catalyses the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic/gluconeogenic pathways. There are two main types of phosphoglycerate mutase: 2,3-bisphosphoglycerate dependent and 2,3-bisphosphoglycerate independent. The enzyme from Saccharomyces cerevisiae has been extensively studied: the high resolution crystal structure of this tetrameric enzyme, subunit Mr 27,000, is known (Winn et al., 1981), the amino acid sequence has been determined (Fothergill and Harkins, 1982) and the gene encoding the enzyme has been isolated and sequenced (White and Fothergill-Gilmore, 1988). Phosphoglycerate mutase from the fission yeast, Schizosaccharomyces pombe, has been purified and partially characterised (Price et al., 1985; Johnson and Price, 1987). It is monomeric, of Mr 23,000, and the sequences of a number of peptides produced by digestion of this enzyme have been determined (Fothergill and Dunbar. unpublished). Alignment of these sequenced peptides with the sequence of S.cerevisiae phosphoglycerate mutase shows 40% identity and the conservation of a number of residues which are known to be essential to the activity of the S. cerevisia enzyme e. g. His-8, Arg-7, Ser-11. Thr-20 and Arg-59. Attempts were made to isolate and sequence the gene encoding S. pombe phosphoglycerate mutase. The S.cerevisiae phosphoglycerate mutase gene failed to detect gene sequence homologies in the S.pombe genome. An oligonucleotide, designed against part of the S.pombe phosphoglycerate mutase sequence (a stretch which was not homologous to the S. cerevisiae sequence) also failed to detect sequence homologies in the S.pombe genome. Thus under the conditions used, neither the S. cerevisiae gene nor the degenerate oligonucleotide appeared to be a suitable molecular probe to screen the S.pombe cDNA expression library in λgt11 (which was synthesised by V. Simanis). A polyclonal antibody against S.pombe phosphoglycerate mutase was prepared and used to screen the S. pombe cDNA expression library. A number of small identical clones were isolated and sequenced. the cDNA inserts encoded 69 residues and part of this sequence was similar to part of the sequence of phosphoglycerate mutase from other sources. Part of the sequence was also similar to a stretch of fructose-2,6-bisphosphatase sequence (fructose-2,6-bisphosphatase appears to be divergently related to phosphoglycerate mutase, Pilkis et al., 1987). A purification scheme for phosphoglycerate mutase from the prokaryote, Streptomyces coelicolor, has been devised. The N-terminal sequence of this enzyme was determined and confirmed that the gene isolated and sequenced by Peter White, encoded phosphoglycerate mutase from S. coelicolor. The enzyme was shown to be a tetramer with a subunit Mr of 29,000. S. coelicolor phosphoglycerate mutase was also shown to be partially 2,3-bisphosphoglycerate dependent.