The overlapping genes encoding phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) from the hyperthermophilic archaeon Sulfolobus solfataricus have been cloned and sequenced. PCR primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the y g k gene. This was then 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 pgk gene and the gap gene immediately downstream. Unexpectedly, the pgk and gap genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene. Evidence that the two genes are co-transcribed was obtained by Northern-blot analysis. The S. soljataricus PGK amino acid sequence shows 43 % and 45 % identity to the PGK sequences of the Archaea Methanobacterium hryantii and Methanothermus fervidus, respectively. High level expression of the S. solfatciricus PGK and GraP-DH in Escherichia coli was achieved, with heat treatment at 80°C proving an effective first step in the purification of these recombinant enzymes from extracts of the E. coli host. Purified recombinant S. solfuturicus PGK and GraP-DH showed half lives of 39 min and 17 h, respectively, at 80°C. Unlike bacterial GraP-DH enzymes, S. solfaturicus GraP-DH was able to use both NAD' and NADP + as cofactors, but exhibited a marked preference for NADP + .Keywords: Su&hhus ; archaea; glycolysis ; phosphoglycerate ; glyceraldehyde.Phosphoglycerate kinase (PGK) catalyses the reversible transfer of a phosphoryl group from 1,3-diphosphoglycerate to ADP to give 3-phosphoglycerate and ATP. This enzyme occurs universally throughout the bacterial, eucaryal, and archaeal kingdoms. Although glucose metabolism in the thermophilic Archaea Sulfolobus and Thermoplasma has been shown to take place via a non-phosphorylated Entner-Doudoroff pathway not involving PGK [I], it appears that Pyrococcus furiosus and other Archaea still possess a PGK that is involved in gluconeogenesis [2].All PGK enzymes, except one form in the hyperthermophilic bacterium Tlzermotogn muritirna, are monomeric with molecular masses of approximately 44 kDa. The pgk gene from 7: maritima was recently reported to encode a 43-kDa PGK and a 70-kDa PGK-triosephosphate isomerase fusion protein, produced by a translational frameshift between the pgk gene and the overlapping tpi gene located downstream 131. Sequences of the PGK enzymes of two Archaea, Metlzanothermus fervidus and MethaCorrespndence to P. W. Piper,
Recombinant Sulfolobus solfataricus glyceraldehyde-3-phosphate dehydrogenase has been purified and found to be a tetramer of 148 kDa. The enzyme shows dual cofactor specificity and uses NADP+ in preference to NAD+. The sequence has been compared with other GAPDH proteins including those from other archaeal sources. The purified protein has been crystallized from ammonium sulfate to produce crystals that diffract to 2.4 A with a space group of P43212 or P41212. A native data set has been collected to 2.4 A using synchrotron radiation and cryocooling.
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