Structural and Enzymatic Investigation of the Sulfolobus solfataricus Uridylate Kinase Shows Competitive UTP Inhibition and the Lack of GTP Stimulation ²,‡ Kristine Steen Jensen, § Eva Johansson, |,⊥,# and Kaj Frank Jensen* ,§ Department of Biological Chemistry, Institute of Molecular Biology, UniVersity of Copenhagen, SølVgade 83H, DK-1307 Copenhagen K, Denmark, Centre for Crystallographic Studies, Department of Chemistry, UniVersity of Copenhagen, UniVersitetsparken 5, DK-2100 Copenhagen Ø, Denmark, and European Synchrotron Radiation Facility, BP220 F38043 Grenoble Cedex, France ReceiVed August 31, 2006; ReVised Manuscript ReceiVed December 8, 2006 ABSTRACT: The pyrH gene encoding uridylate kinase (UMPK) from the extreme thermoacidophilic archaeon Sulfolobus solfataricus was cloned and expressed in Escherichia coli, and the enzyme (SsUMPK) was purified. Size exclusion chromatography and sedimentation experiments showed that the oligomeric state in solution is hexameric. SsUMPK shows maximum catalytic rate at pH 7.0, and variation of pH only influences the turnover number. Catalysis proceeds by a sequential reaction mechanism of random order and depends on a divalent cation. The enzyme exhibits high substrate specificity toward UMP and ATP and is inhibited by UTP, whereas CTP and GTP do not influence activity. UTP binds to the enzyme with a sigmoid binding curve, whereas GTP does not bind. The crystal structure of SsUMPK was determined for three different complexes, a ternary complex with UMP and the nonhydrolyzable ATP analogue ,γ- methylene-ATP, a complex with UMP, and a complex with UTP to 2.1, 2.2, and 2.8 Å resolution, respectively. One UTP molecule was bound in the acceptor site per subunit, leading to the exclusion of both substrates from the active site. In all cases, SsUMPK crystallized as a hexamer with the main fold shared with other prokaryotic UMPKs. Similar to UMPK from Pyrococcus furiosus, SsUMPK has an active site enclosing loop. This loop was only ordered in one subunit in the ternary complex, which also contained an unusual arrangement of ligands (possibly a dinucleotide) in the active site and an altered orientation of the catalytic residue Arg48 relative to the other five subunits of the hexamer. Uridylate kinase (UMPK 1 ) plays a key role in pyrimidine biosynthesis by catalyzing the ATP dependent phosphory- lation of UMP, thus forming UDP. UDP may be further phosphorylated to UTP, which is a substrate for RNA polymerase, a precursor for CTP synthesis, and a cofactor in sugar metabolism. Another fate of UDP is the reduction to dUDP as a step in the pathway for the synthesis of dTTP and dCTP. The UMPKs of bacterial origin differ considerably from the UMPKs of eukaryotic origin. In general, the eukaryotic enzymes have a broader substrate specificity, being able to phosphorylate both UMP and CMP, than the enzymes of bacterial origin, which show a strong preference for UMP (1). Another difference between the two classes of enzymes is that the UMPKs of eukaryotic origin are monomeric proteins with sizes around 25 kDa, whereas the UMPKs of bacterial origin are hexamers of identical polypeptides with sizes around 25 kDa (1, 2). The recent determination of the crystal structures of several UMPKs of prokaryotic origin have shown that the overall fold of the prokaryotic monomer differs significantly from the fold of the enzymes of eukaryotic origin (3, 4). In general, the catalytic mechanism for nucleoside mono- phosphate kinases is sequential with no obligatory order for binding substrates and departure of products (1). The phosphoryl transfer from ATP to UMP is believed to be of mainly associative nature with a penta-coordinated transition state (5), although the enzyme’s ways of stabilizing the transition state and the geometry of the transition state remain unclear (1, 6). The regulation of the activity of bacterial UMPKs is complex, involving feedback inhibition by UTP and activa- tion by GTP (2, 7, 8). The mechanism by which GTP ² This work was supported by grants from the Danish National Science Research Council and the European Community-Research Infrastructure Action under the FP6 program “Structuring the European Research Area”. We also acknowledge the contribution of the Danish Natural Science Council to DANSYNC. ‡ The coordinates (and structure factors) have been deposited in the Protein Data Bank with accession codes 2j4j (r2j4jsf), 2j4k (r2j4ksf), and 2j4l (r2j4lsf) for the S. solfataricus uridylate kinase in complex with UMP and AMPPCP, UMP, and UTP, respectively. * To whom correspondence should be addressed. Tel: +45 3532 2020. Fax: +45 3532 2040. E-mail: kfj@mermaid.molbio.ku.dk. § Department of Biological Chemistry, University of Copenhagen. | Center for Crystallographic Studies, University of Copenhagen. ⊥ European Synchrotron Radiation Facility. # Present address: Diabetes Protein Engineering, Novo Nordisk A/S, Novo Alle ´ 6, DK-2880 Bagsværd, Denmark. 1 Abbreviations: UMPK, uridine 5′-monophosphate kinase (EC 2.7.4.22); SsUMPK, S. solfataricus uridine 5′-monophosphate kinase; SsUMPK-UMP, SsUMPK in complex with uridine 5′-monophosphate; SsUMPK-UMP-AMPPCP, SsUMPK in complex with uridine 5′- monophosphate and ,γ-methylene adenosine 5′-triphosphate; SsUMPK- UTP, SsUMPK in complex with uridine 5′-triphosphate; buffer A, 25 mM Tris-HCl and 0.1 mM EDTA at pH 7.6; PCR, polymerase chain reaction; PEI-plates, poly(ethyleneimine)-impregnated cellulose thin- layer plates on plastic sheets. 2745 Biochemistry 2007, 46, 2745-2757 10.1021/bi0618159 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/13/2007