Phosphoenolpyruvate: Sugar Phosphotransferase System from the Hyperthermophilic Thermoanaerobacter tengcongensis

Abstract

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.

Conformational Selection and Substrate Binding Regulate the Monomer/Dimer Equilibrium of the C-terminal domain of Escherichia coli Enzyme I

“…NMR structural characterization shows that in solution EIC adopts the same tertiary fold and dimeric structure as that observed in the various crystal structures of EI (15,16,26) and EIC (27,28). NMR 15 N relaxation dispersion data also reveal the existence of conformational exchange on the submillisecond time scale in the region of the PEP binding and at the ␤3␣3 turn.…”

“…These large-scale conformational changes permit transfer of the phosphoryl group from PEP bound to the EIC domain to His 189 located on EIN in the conformation found in the trapped phosphorylated intermediate (15), and subsequent transfer of the phosphoryl group on His 189 to HPr in the conformation found in the structures of free EI and the EI-HPr complex where the structure of the EIN domain is identical to that of the isolated EIN domain (17). In addition, crystal structures of the isolated EIC domain from Thermoanaerobacter tengcongensis in the free form (27) and in complexes with PEP and pyruvate (28) have been obtained. Although the structure of the EIC domain is the same in intact EI and the isolated EIC domain, spectroscopic and kinetic investigations have suggested that in solution the EIC domain may be present as an ensemble of different conformations that are not apparent in the crystal structures (14).…”

“…Solution Tertiary Structure of Free E. coli EIC-Crystallographic data show that E. coli EIC is a ϳ 70-kDa dimer exhibiting an (␣/␤) 8 ) in the active site but leaves the overall tertiary fold and quaternary structure unperturbed (28). By way of contrast, sedimentation velocity and thermal denaturation exper-iments have shown that the addition of PEP stabilizes the protein fold and decreases the equilibrium dissociation constant for dimerization (14).…”

“…B, structural model for the E. coli EIC-PEP complex. The model was obtained by rigid-body least-square fitting of the backbone atoms of the x-ray structure of the EIC-PEP complex from T. tengcongensis (28) onto the EIC coordinates of the x-ray structure of phosphorylated EI from E. coli (15). The fitting was carried out using residues 261-333 and 367-570, and the C␣ rms difference is 1.…”

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

“…However, the crystal structure of EIC from Thermoanaerobacter tengcongensis recently showed a conformational change of Phe354 upon binding to PEP. 19 As Phe354 is conserved in EIC of E. coli, it likely contributed to the change in the CD and NMR spectra in this study. In addition, Tyr459 and Tyr474 are the nearest neighboring aromatic residues to the PEP binding site, which may have contributed to the spectroscopic changes observed in this study.…”