A Flexible Loop at the Dimer Interface is a Part of the Active Site of the Adjacent Monomer of Escherichia coli Orotate Phosphoribosyltransferase †,‡ Anette Henriksen,* ,§ Nushin Aghajari, § Kaj Frank Jensen, | and Michael Gajhede § Center for Crystallographic Studies, UniVersity of Copenhagen, UniVersitetsparken 5, DK-2100 KøbenhaVn Ø, Denmark, and Center for Enzyme Research, UniVersity of Copenhagen, SølVgade 83 H, DK-1307 KøbenhaVn K, Denmark ReceiVed September 18, 1995; ReVised Manuscript ReceiVed January 15, 1996 X ABSTRACT: Orotate phosphoribosyltransferase (OPRTase) is involved in the biosynthesis of pyrimidine nucleotides. R-D-ribosyldiphosphate 5-phosphate (PRPP) and orotate are utilized to form pyrophosphate and orotidine 5′-monophosphate (OMP) in the presence of divalent cations, preferably Mg 2+ . OMP is thereafter converted to uridine 5′-monophosphate by OMP decarboxylase. We have determined the 2.4 Å structure of Escherichia coli OPRTase, ligated with sulfate, by molecular replacement and refined the structure to an R-factor of 18.3% for all data. In the structure of the E. coli enzyme we have determined the fold of a flexible loop region with a highly conserved amino acid sequence among OPRTases, a region known to take part in catalysis. The structure of this region was not determined in the model used for molecular replacement, and it involves interactions at the dimer interface through a bound sulfate ion. Crystalline E. coli OPRTase is a homodimer, with sulfate ions inhibiting enzyme activity bound in the dimer interface close to the flexible loop region. Although this loop is very close in space to the sulfate binding site, and sulfate is found in both interfaces of the homodimer, the loop structure is only traceable in one monomer. We expect that the mobility of this loop is important for catalysis, and, on the basis of the reported structure and the structure of Salmonella typhimurium OPRTase‚OMP, we propose that the movement of this loop in association with the movement of OMP is vital to catalysis. Apart from the flexible loop region and a solvent-exposed loop (residues 158-164), the most significant differences in structure between S. typhimurium OPRTase‚OMP and E. coli OPRTase are found in the substrate binding regions: the 5′-phosphate binding region (residues 120-131), the binding region for the orotate part of OMP (residues 25-27), and the pyrophosphate binding region (residues 71-73). Orotate phosphoribosyltransferase (OPRTase) provides the pathway for de noVo biosynthesis of pyrimidine nucleotides by catalyzing the Mg 2+ dependent formation of orotidine 5′- monophosphate (OMP), the pyrimidine nucleotide from which uridine 5′-monophosphate is synthesized (Figure 1) (Musick, 1981). Other phosporibosyltransferases (PRTases) are involved in synthesis or salvage of purine and pyrimidine nucleotides and also in the synthesis of the aromatic amino acids histidine and tryptophan and the pyridine coenzymes NAD and NADP (Musick, 1981, Jensen, 1983). The PRTases all transfer a ribosyl phosphate group from R-D- ribosyldiphosphate 5-phosphate (PRPP) with the C1′ of ribose as the target position. A motif of 12 amino acid residues also found in PRPP synthetases has been proposed to represent a common PRPP binding motif (Hershey & Taylor, 1986; Hove-Jensen et al., 1986). This short sequence is the only well-conserved sequence observed in the group of PRTases and structurally it represents a strand-loop- helix structure. In two of the known three-dimensional structures of PRTases, OPRTase‚OMP (Scapin et al., 1994) and hypox- anthine-guanine-PRTase(HGPRTase)‚GMP (Eads et al., 1994), a homologous stretch of amino acid residues showed very poor density. This loop was left structurally non-determined in OPRTase, while a tentative fitting of the sequence to the electron density was made in one HGPRTase monomer, showing a loop extending into the solvent. The sequence of this very flexible loop is highly conserved among OPRTases, and residues from the loop are important for catalysis (Grubmeyer et al., 1993; Ozturk et al., 1995a,b). The third published PRTase structure, glutamine-PRPP- amidotransferase‚AMP (amido-PRTase) belongs to the glutamine amidotransferase enzyme family as well (Smith et al., 1994). This enzyme has an even longer extended structure in a similar position, making contacts between neighboring subunits. In amido-PRTase the region has been proposed to be involved in feedback regulation of enzyme activity (Smith et al., 1994). The reaction mechanism of OPRTases is still a subject of discussion. The transferase reaction proceeds with inversion at the anomeric carbon, and an oxocarbonium-like transition state has been proposed (Bhatia et al., 1990; Goitein et al., 1978) as well as an S N 1-like mechanism (Goitein et al., 1978). We have initiated structural investigations of E. coli OPRTase to establish the structural basis for the PRTase function in this quite small non-allosterically controlled enzyme. † This work was funded by the Danish National Research Foundation. ‡ Coordinates for this structure have been deposited in the Brookhaven Protein Data Bank. The access code is 1oro. * Author to whom correspondence should be addressed. § Center for Crystallographic studies. | Center for Enzyme Research. X Abstract published in AdVance ACS Abstracts, February 15, 1996. 1 Abbreviations: OPRTase, orotate phosphoribosyltransferase; OMP, orotidine 5′-monophosphate; PRPP, R-D-ribosyldiphosphate 5-phos- phate; AMP, adenosine 5′-monophosphate; GMP, guanosine 5′- monophosphate; PRTase, phosphoribosyltransferase; HPRTase, hy- poxanthine-guanine phosphoribosyltransferase; amido-PRTase, glutamine PRPP amidotransferase; Fo, measured structure factor amplitude; Fc, calculated structure factor amplitude; (Fo - Fc)Rcalc, electron density calculated with (|Fo|-|Fc|) coefficients and phases calculated from the coordinates; (2Fo-Fc)Rcalc, electron density calculated with (2|Fo|- |Fc|) coefficients and phases calculated from the coordinates; Rcryst, ∑||Fo|-|Fc||/∑|Fo|; Rmerge, ∑∑〈I〉 - I/∑∑I. 3803 Biochemistry 1996, 35, 3803-3809 0006-2960/96/0435-3803$12.00/0 © 1996 American Chemical Society