Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus.
Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-alpha-1-diphosphate (PRPP) and uracil to uridine monophosphate (UMP) and diphosphate (PP(i)). The tetrameric enzyme from Sulfolobus solfataricus has a unique type of allosteric regulation by cytidine triphosphate (CTP) and guanosine triphosphate (GTP). Here we report two structures of the activated state in complex with GTP. One structure (refined at 2.8-A resolution) contains PRPP in all active sites, while the other structure (refined at 2.9-A resolution) has PRPP in two sites and the hydrolysis products, ribose-5-phosphate and PP(i), in the other sites. Combined with three existing structures of uracil phosphoribosyltransferase in complex with UMP and the allosteric inhibitor cytidine triphosphate (CTP), these structures provide valuable insight into the mechanism of allosteric transition from inhibited to active enzyme. The regulatory triphosphates bind at a site in the center of the tetramer in a different manner and change the quaternary arrangement. Both effectors contact Pro94 at the beginning of a long beta-strand in the dimer interface, which extends into a flexible loop over the active site. In the GTP-bound state, two flexible loop residues, Tyr123 and Lys125, bind the PP(i) moiety of PRPP in the neighboring subunit and contribute to catalysis, while in the inhibited state, they contribute to the configuration of the active site for UMP rather than PRPP binding. The C-terminal Gly216 participates in a hydrogen-bond network in the dimer interface that stabilizes the inhibited, but not the activated, state. Tagging the C-terminus with additional amino acids generates an endogenously activated enzyme that binds GTP without effects on activity.
Christoffersen
Stig
S
Department of Biology, University of Copenhagen, Biocenter, Copenhagen N, Denmark.
Kadziola
Anders
A
Johansson
Eva
E
Rasmussen
Michael
M
Willemoës
Martin
M
Jensen
Kaj Frank
KF
D000494:Allosteric Regulation; D019843:Archaeal Proteins; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D014542:Uridine Monophosphate / Q000737:chemistry / Q000378:metabolism
D019843:Archaeal Proteins; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D014542:Uridine Monophosphate; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase