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.
Department for Chemistry and Biochemistry, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland.
Vera
V
Navdaeva
Andreas
A
Zurbriggen
Sandro
S
Waltersperger
Philipp
P
Schneider
Anselm E
AE
Oberholzer
Priska
P
Bähler
Christoph
C
Bächler
Andreas
A
Grieder
Ulrich
U
Baumann
Bernhard
B
Erni
D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology; D006358:Hot Temperature; D008954:Models, Biological; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D046968:Thermoanaerobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D050505:Mutant Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System