TY - JOUR

T1 - Structural and kinetic studies of the allosteric transition in Sulfolobus solfataricus uracil phosphoribosyltransferase: Permanent activation by engineering of the C-terminus

AU - Christoffersen, Stig

AU - Kadziola, Anders

AU - Johansson, Eva

AU - Rasmussen, Michael

AU - Willemoës, Martin

AU - Jensen, Kaj Frank

N1 - Keywords: nucleotide biosynthesis; UMP; GTP; CTP; Archaea; enzyme regulation

PY - 2009

Y1 - 2009

N2 - Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-a-1-diphosphate (PRPP) and uracil to uridine monophosphate(UMP) and diphosphate (PPi). The tetrameric enzyme from Sulfolobussolfataricus has a unique type of allosteric regulation by cytidinetriphosphate (CTP) and guanosine triphosphate (GTP). Here we reporttwo structures of the activated state in complex with GTP. One structure(refined at 2.8-Å resolution) contains PRPP in all active sites, while the otherstructure (refined at 2.9-Å resolution) has PRPP in two sites and thehydrolysis products, ribose-5-phosphate and PPi, in the other sites.Combined with three existing structures of uracil phosphoribosyltransferasein complex with UMP and the allosteric inhibitor cytidine triphosphate(CTP), these structures provide valuable insight into the mechanism ofallosteric transition from inhibited to active enzyme. The regulatory triphosphatesbind at a site in the center of the tetramer in a different mannerand change the quaternary arrangement. Both effectors contact Pro94 at thebeginning of a long ß-strand in the dimer interface, which extends into aflexible loop over the active site. In the GTP-bound state, two flexible loopresidues, Tyr123 and Lys125, bind the PPi moiety of PRPP in the neighboringsubunit and contribute to catalysis, while in the inhibited state, theycontribute to the configuration of the active site for UMP rather than PRPPbinding. The C-terminal Gly216 participates in a hydrogen-bond network inthe dimer interface that stabilizes the inhibited, but not the activated, state.Tagging the C-terminus with additional amino acids generates anendogenously activated enzyme that binds GTP without effects on activity.

AB - Uracil phosphoribosyltransferase catalyzes the conversion of 5-phosphoribosyl-a-1-diphosphate (PRPP) and uracil to uridine monophosphate(UMP) and diphosphate (PPi). The tetrameric enzyme from Sulfolobussolfataricus has a unique type of allosteric regulation by cytidinetriphosphate (CTP) and guanosine triphosphate (GTP). Here we reporttwo structures of the activated state in complex with GTP. One structure(refined at 2.8-Å resolution) contains PRPP in all active sites, while the otherstructure (refined at 2.9-Å resolution) has PRPP in two sites and thehydrolysis products, ribose-5-phosphate and PPi, in the other sites.Combined with three existing structures of uracil phosphoribosyltransferasein complex with UMP and the allosteric inhibitor cytidine triphosphate(CTP), these structures provide valuable insight into the mechanism ofallosteric transition from inhibited to active enzyme. The regulatory triphosphatesbind at a site in the center of the tetramer in a different mannerand change the quaternary arrangement. Both effectors contact Pro94 at thebeginning of a long ß-strand in the dimer interface, which extends into aflexible loop over the active site. In the GTP-bound state, two flexible loopresidues, Tyr123 and Lys125, bind the PPi moiety of PRPP in the neighboringsubunit and contribute to catalysis, while in the inhibited state, theycontribute to the configuration of the active site for UMP rather than PRPPbinding. The C-terminal Gly216 participates in a hydrogen-bond network inthe dimer interface that stabilizes the inhibited, but not the activated, state.Tagging the C-terminus with additional amino acids generates anendogenously activated enzyme that binds GTP without effects on activity.

U2 - 10.1016/j.jmb.2009.08.019

DO - 10.1016/j.jmb.2009.08.019

M3 - Journal article

C2 - 19683539

VL - 393

SP - 464

EP - 477

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

IS - 2

ER -