Atomic structure at 2.5 Å resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice

Abstract

Received 18 april 1995 homology to the sequence of E. coli PNP -the sequence simiAbstract Uridine phosphorylase from E. coil (Upase) has been larity being 49% and the sequence identity 25% [9]. However crystallized using vapor diffusion technique in a new monoclinic the sequence homology between Upase and other phosphorycrystal form. The structure was determined by the molecular lases is very poor. The comparison of the three-dimensional replacement method at 2.5 ~. resolution. The coordinates of the trigonal crystal form were used as a starting model and the structures of Upase and other phosphorylases has shown that refinement by the program XPLOR led to the R-factor of 18.6%.the structure of Upase and human PNP are rather similar but The amino acid fold of the protein was found to be the same as there is no any similarity between Upase and thymidine that in the trigonal crystals. The positions of flexible regions were phosphorylase structures. The study of E. coli PNP is in prorefined. The conclusion about the involvement in the active site gress and obviously its structure must be similar to that of is in good agreement with the results of the biochemical experiUpase. The Upase three-dimensional structure has been solved ments, at 3.0 ~ resolution using the data from trigonal crystals described previously [9]. This structure contains some disordered Key words: Uridine phosphorylase; E. coli; X-ray structure regions and little is known about groups involved in catalysis and/or binding at the Upase active site [2,10 12]. IntroductionIn this paper we report results of an X-ray investigation of a monoclinic form at 2.5 ~ resolution and the interpretation of the data of the selective chemical modification of the essential Uridine phosphorylase (EC 2.4.2.3; Upase) from E. coli catamino acid residues. alyzes the reversible phosphorolysis of uridine with the formation of riboso-l-phosphate and uracil [1,2]. The protein is involved in degradation of pyrimidine nucleosides and their util-2. Materials and methods ization as carbon and energy sources in E. coli cells. Upase 2.1. Crystallization along with purine nucleoside phosphorylase (PNP) and thymid-The uridine phosphorylase was crystallized in different crystal forms ine phosphorylase, belongs to the class of nucleoside by the vapor diffusion technique at room temperature [13,14]. The phosphorylases. Uridine phosphorylase has been identified as monoclinic form was obtained in sitting drops of 0.05 M Tris-HC1 the enzyme which is responsible for the cleavage of some pyrimbuffer at pH 7.3 containing 10-12 mg/ml of the protein and 4-6% PEG (mol.wt. 4,000). The equilibrium solution consisted of 0.1 M Tris-mal/ idine nucleoside analogs possessing antitumor activity. ConverNaOH pH 5.91 5.96, 20-25% PEG (mol.wt. 4,000) and 0.04% sodium sion of 5-fluorouridine (FUR) and 5-fluorodeoxyuridine azide. The crystals reached the size of 0.7 × 0.3 x 0.5 mm after 4-6 (FUdR) to 5-fluorouracil (5FU) by Upase required the usage weeks. These crystals belong to monoclinic space group P2~...

Transition State Analogue Inhibitors of Purine Nucleoside Phosphorylase from Plasmodium falciparum

“…2) identified residues with specific functions in catalysis but only residues near the active site (35). These residues are no more conserved in P. falciparum PNP than they are in the bacterial PNPs.…”

Three-dimensional Structure of a Hyperthermophilic 5′-Deoxy-5′-methylthioadenosine Phosphorylase from Sulfolobus solfataricus

“…5a. The phosphatebinding sites of E. coli PNP (7,14) and E. coli (15) uridine phosphorylase use the same functional groups and the same binding geometry as SsMTAP, except that Thr 89 is replaced by serine in these enzymes. In contrast, the phosphate-binding sites of trimeric enzymes in the PNP family are similar to each other (8, 13) but distinctly different from those of the hexameric enzymes.…”

“…Met 181 provides another key interaction, packing against the hydrophobic face of the ribose, Phe 160 and the purine base. His 5 , Met 181 , and Glu 182 are highly conserved among the hexameric members of the PNP family (7,15). A methionine residue equivalent to Met 181 has been identified in all known nucleoside phosphorylase structures (7,8,(11)(12)(13)(14)28).…”

“…The crystal structure of E. coli PNP has also been described and represents an example from the hexameric class of enzymes (7,14). The structure of uridine phosphorylase from E. coli (15) represents a second example of the hexameric class. The structures of thymidine phosphorylase from E. coli (16,17) and pyrimidinenucleoside phosphorylase from Bacillus stearothermophilus (18) show that these enzymes belong to a separate structural family.…”

Behavior of uridine phosphorylase from Escherichia coli K‐12 in hydrated reversed micelles of surfactant in organic solvent

“…The X-ray data testifies that hexamer is formed from three dimers, two dimers in each trimer being connected by non-crystallographic symmetry axis of the second order (20,21).…”