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 homogeneity, and its coding gene was cloned and expressed in Escherichia coli. It represents a homomeric tetramer with a molecular mass of 49 kDa per subunit. PK exhibits positive binding cooperativity with respect to phosphoenolpyruvate and metal ions such as Mg(2+) and Mn(2+). Heterotropic effects, as commonly found for PKs from bacterial and eucaryal sources, could not be detected. The enzyme does not depend on K(+) ions. Heterotrophically grown cells exhibit specific activity of PK four times higher than autotrophically grown cells. Since the mRNA level of the PK coding gene is also accordingly higher in heterotrophic cells, we conclude that the PK activity is adjusted to growth conditions mainly on the transcript level. The enzymic properties of the PK and the regulation of its expression are discussed with respect to the physiological framework given by the T. tenax-specific variant of the Embden-Meyerhof-Parnas pathway. T. tenax PK shows moderate overall sequence similarity (25 to 40% identity) to its bacterial and eucaryal pendants. Phylogenetic analyses of the known PK sequences result in a dichotomic tree topology that divides the enzymes into two major PK clusters, probably diverged by an early gene duplication event. The phylogenetic divergence is paralleled by a striking phenotypic differentiation of PKs: PKs of cluster I, which occur in eucaryal cytoplasm, some gamma proteobacteria, and low-GC gram-positive bacteria, are only active in the presence of fructose-1,6-bisphosphate or other phosphorylated sugars, whereas PKs of cluster II, found in various bacterial phyla, plastids, and in Archaea, show activity without effectors but are commonly regulated by the energy charge of the cell.
Schramm
A
A
Department of Microbiology, Universität GH Essen, D-45117 Essen, Germany.
Siebers
B
B
Tjaden
B
B
Brinkmann
H
H
Hensel
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D003001:Cloning, Molecular; D019848:Gene Expression Regulation, Archaeal / Q000235:genetics; D020131:Genes, Duplicate / Q000235:genetics; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019642:RNA, Archaeal / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019624:Thermoproteaceae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D002413:Cations, Divalent; D019642:RNA, Archaeal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D008274:Magnesium