Dimeric 3‐Phosphoglycerate Kinases from Hyperthermophilic Archaea

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Abstract
The gene coding for the 3‐phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical Mr of 46195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3‐phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19–25]. By comparing the 3‐phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde‐3‐phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329–4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases. To study the phenotypic properties of the 3‐phosphoglycerate kinases from thermophilic Archaea in more detail, the 3‐phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects, (a) The 3‐phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3‐phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+. For the P. woesei enzyme, non‐cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M. fervidus 3‐phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non‐cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd= 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced. At the optimum growth temperature of P. woesei (100°C) and M. fervidus (83°C), the 3‐phosphoglycerate kinases show half‐lives of inactivation of only 28 min and 44 min, respectively. Potassium salts of polyvalent anions stabilize both 3‐phosphoglycerate kinases, like other enzymes from these organisms. In the case of M. fervidus 3‐phosphoglycerate kinase, the dominant low‐molecular‐mass compound cyclic d‐glycerate 2,3‐bisphosphate found in vivo [Hensel, R. & König, H. (1988) FEMS Microbiol. Lett. 19, 325–333] is the most effective.

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