Crystal Structure of the Complex of UMP/CMP Kinase from Dictyostelium discoideum and the Bisubstrate Inhibitor P¹-(5‘-Adenosyl) P⁵-(5‘-Uridyl) Pentaphosphate (UP₅A) and Mg²⁺ at 2.2 Å:  Implications for Water-Mediated Specificity

Abstract

The three-dimensional structure of the UMP/CMP kinase (UK) from the slime mold Dictyostelium discoideum complexed with the specific and asymmetric bisubstrate inhibitor P1-(5'-adenosyl) P5-(5'-uridyl) pentaphosphate (UP5A) has been determined at a resolution of 2.2 A. The structure of the enzyme, which has up to 41% sequence homology with known adenylate kinases (AK), represents a closed conformation with the flexible monophosphate binding domain (NMP site) being closed over the uridyl moiety of the dinucleotide. Two water molecules were found within hydrogen-bonding distance to the uracil base. The key residue for the positioning and stabilization of those water molecules appears to be asparagine 97, a residue that is highly specific for AK-homologous UMP kinases, but is almost invariably a glutamine in adenylate kinases. Other residues in this region are highly conserved among AK-related NMP kinases. The catalytic Mg2+ ion is coordinated with octahedral geometry to four water molecules and two oxygens of the phosphate chain of UP5A but has no direct interactions with the protein. The comparison of the geometry of the UKdicty.UP5A.Mg2+ complex with the previously reported structure of the UKyeast.ADP.ADP complex [Müller-Dieckmann & Schulz (1994) J. Mol. Biol. 236, 361-367] suggests that UP5A in our structure mimics an ADP.Mg.UDP biproduct inhibitor rather than an ATP. MG.UMP bisubstrate inhibitor.

Purine and Pyrimidine Nucleotide Synthesis and Metabolism

“…The plant UMPK is insensitive to GTP and UTP. Eukaryotic UMPKs all share a conserved glycine-rich sequence in their N-terminal regions which is referred to as the phosphate-binding loop and may play a role in ATP binding and/or enzyme catalysis (MullerDieckmann and Schulz, 1994, Muller-Dieckmann and Schulz, 1995, Scheffzek et al, 1996. Site-specific mutations within this glycine-rich conserved region of the Arabidopsis enzyme resulted in significant changes in its catalytic activity.…”

Substrate-induced Conformational Changes in Human UMP/CMP Kinase

“…Of special importance is 3TC (␤-L-2Ј,3Ј-dideoxy-3Ј-thiacytidine) used in the treatment of human immunodeficiency virus infection. However, prior to this work, our structural understanding of this important enzyme was limited to the yeast uridylate kinase (6) (50% sequence identity to the human enzyme) and the UMP/CMP kinase from the slime mold Dictyostelium discoideum (52% identity) (7). Here we present the structure of the human UMP/CMP kinase in its apo conformation.…”

Use of pyrimidine pathway to produce mutant plants that fail to respond to wound induction

“…The crystal structures of UMP kinases from yeast and D. discoideum suggested that this consensus sequence may play a role in ATP binding and/or enzyme catalysis (Miiller-Deickmann andSchulz, 1994 andScheffzek et al, 1996). The sitedirected mutagenesis studies in this report support this suggestion.…”

“…Other conserved sequences are also necessary. In fact, in the structure of the enzyme from yeast (Muller-Dieckmann andSchulz, 1994 and or D. discoideum (Scheffzek et al. 1996), there were many other conserved residues involved in ATP-binding in the active site of the enzyme.…”

“…The crystal structures of UMP kinases from S. cerevisiae and D. discoideum have been solved Schulz 1994 andScheffzek et al, 1996). The crystal structure of UMP/CMP kinase from D. discoideum complexed with a bisubstrate inhibitor P'-(5'-adenosyl) P^-(5'-uridyl) pentaphosphate (UP5A) with a resolution of 2.2 A showed that the globular enzyme consisted of eight a-helixes that surround five parallel p-sheets.…”