Phosphoenolpyruvate: sugar phosphotransferase system from the hyperthermophilic Thermoanaerobacter tengcongensis.

Thermoanaerobacter tengcongensis is a thermophilic eubacterium that has a phosphoenolpyruvate (PEP) sugar phosphotransferase system (PTS) of 22 proteins. The general PTS proteins, enzyme I and HPr, and the transporters for N-acetylglucosamine (EIICB(GlcNAc)) and fructose (EIIBC(Fru)) have thermal unfolding transitions at ∼90 °C and a temperature optimum for in vitro sugar phosphotransferase activity of 65 °C. The phosphocysteine of a EIICB(GlcNAc) mutant is unusually stable at room temperature with a t(1/2) of 60 h. The PEP binding C-terminal domain of enzyme I (EIC) forms a metastable covalent adduct with PEP at 65 °C. Crystallization of this adduct afforded the 1.68 Å resolution structure of EIC with a molecule of pyruvate in the active site. We also report the 1.83 Å crystal structure of the EIC-PEP complex. The comparison of the two structures with the apo form and with full-length EI shows differences between the active site side chain conformations of the PEP and pyruvate states but not between the pyruvate and apo states. In the presence of PEP, Arg465 forms a salt bridge with the phosphate moiety while Glu504 forms salt bridges with Arg186 and Arg195 of the N-terminal domain of enzyme I (EIN), which stabilizes a conformation appropriate for the in-line transfer of the phosphoryl moiety from PEP to His191. After transfer, Arg465 swings 4.8 Å away to form an alternative salt bridge with the carboxylate of Glu504. Glu504 loses the grip of Arg186 and Arg195, and the EIN domain can swing away to hand on the phosphoryl group to the phosphoryl carrier protein HPr.

Crystal structure of the phosphoenolpyruvate-binding enzyme I-domain from the Thermoanaerobacter tengcongensis PEP: sugar phosphotransferase system (PTS).

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.

From ATP as substrate to ADP as coenzyme: functional evolution of the nucleotide binding subunit of dihydroxyacetone kinases.

Dihydroxyacetone kinases are a family of sequence-related enzymes that utilize either ATP or a protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as a source of high energy phosphate. The PTS is a multicomponent system involved in carbohydrate uptake and control of carbon metabolism in bacteria. Phylogenetic analysis suggests that the PTS-dependent dihydroxyacetone kinases evolved from an ATP-dependent ancestor. Their nucleotide binding subunit, an eight-helix barrel of regular up-down topology, retains ADP as phosphorylation site for the double displacement of phosphate from a phospho-histidine of the PTS protein to dihydroxyacetone. ADP is bound essentially irreversibly with a t((1/2)) of 100 min. Complexation with ADP increases the thermal unfolding temperature of dihydroxyacetone L from 40 (apo-form) to 65 degrees C (holoenzyme). ADP assumes the same role as histidines, cysteines, and aspartic acids in histidine kinases and PTS proteins. This conversion of a substrate binding site into a cofactor binding site reflects a remarkable instance of parsimonious evolution.

Escherichia coli dihydroxyacetone kinase controls gene expression by binding to transcription factor DhaR.

Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize either ATP (in animals, plants, bacteria) or the bacterial phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) as a source of high-energy phosphate. The PTS-dependent kinase of Escherichia coli consists of three subunits: DhaK contains the Dha binding site, DhaL contains ADP as cofactor for the double displacement of phosphate from DhaM to Dha, and DhaM provides a phospho-histidine relay between the PTS and DhaL::ADP. DhaR is a transcription activator belonging to the AAA+ family of enhancer binding proteins. It stimulates transcription of the dhaKLM operon from a sigma70 promoter and autorepresses dhaR transcription. Genetic and biochemical studies indicate that the enzyme subunits DhaL and DhaK act antagonistically as coactivator and corepressor of the transcription activator by mutually exclusive binding to the sensing domain of DhaR. In the presence of Dha, DhaL is dephosphorylated and DhaL::ADP displaces DhaK and stimulates DhaR activity. In the absence of Dha, DhaL::ADP is converted by the PTS to DhaL::ATP, which does not bind to DhaR.