Pyruvate kinase of the hyperthermophilic crenarchaeote Thermoproteus tenax: Physiological role and phylogenetic aspects

Pyruvate kinase (PK; EC 2.7.1.40) of Thermoproteus tenax was purified to ho mogeneity, and its coding gene was cloned and expressed in Escherichia coli . It represents a homomeric tetramer with a molecular mass of 49 kDa per su bunit. PK exhibits positive binding cooperativity with respect to phosphoen olpyruvate and metal ions such as Mg2+ and Mn2+. Heterotropic effects, as c ommonly found for PKs from bacterial and eucaryal sources, could not be det ected. The enzyme does not depend on K+ ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically gro wn cells. Since the mRNA level of the PK coding gene is also accordingly hi gher in heterotrophic cells, we conclude that the PK activity is adjusted t o growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect t o the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequenc e similarity (25 to 40% identity) to its bacterial and eucaryal pendants. P hylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably dive rged by an early gene duplication event. The phylogenetic divergence is par alleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gr ampositive bacteria, are only active in the presence of fructose-1,6-bispho sphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without ef fecters but are commonly regulated by the energy charge of the cell.