Structure and conformation of 3'-O-acetyl-2'-deoxy-5-methoxymethyluridine.

C13H18N2O7, Mr = 314.297, triclinic, P1, a = 6.0321 (4), b = 6.775 (5), c = 9.6699 (7) A, alpha = 76.917 (6), beta = 78.871 (6), gamma = 75.344 (6) degrees, V = 368.54 A3, Z = 1, Dm = 1.43, Dx = 1.416 g cm-3, Cu K alpha radiation (Ni filtered), lambda = 1.5418 A, F(000) = 166, T = 287 K, final conventional R factor = 0.034, wR = 0.044 for 1359 reflections and 268 variables. The structure was solved using the XTAL system. The conformation of the furanose ring is best described as intermediate between 2E and 2(1)T: the pseudorotational parameters are P = 148.9 degrees and tau m = 33.4 degrees. The CH2OH, C(5'), side chain has the g+ conformation, the carbonyl bond of the 3'-acetoxy group is syn to the C(3')-O(3',1) bond on the sugar ring and the glycosidic bond conformation is anti [chi = -137.6 (3) degrees]. The methoxy group of the 5-methoxymethyl substituent is on the same side of the pyrimidine plane as O(4') of the furanose ring. Comparison with 2'-deoxy-5-methoxymethyluridine shows that intermolecular attraction have little effect on the internal conformations of the molecule in the solid state.

Tertiary structure of histidine-containing protein of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

The tertiary structure of the histidine-containing phosphocarrier protein (HPr) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has been determined by x-ray diffraction at 2.8-A resolution. Initially, a partial structure was fitted to the multiple isomorphous replacement map and then least-squares refined by the Konnert/Hendrickson restrained parameter method (Konnert, J. H., and Hendrickson, W. A. (1980) Acta Crystallogr. A36, 344-350) and finally, a subsequent map was computed by use of the phase combination method of Read (Read, R. J. (1986) Acta Crystallogr. A42, 140-149). More of the protein structure was located in the latter map. The procedure of model building, least-squares refinement, and electron density map recalculation was repeated until the tertiary structure of HPr was obtained. The overall structure of HPr consists of four beta-strands, three helical regions, and four beta-turns. At the active center, the His15 imidazole interacts with one oxygen atom of the alpha-carboxyl C terminus of the polypeptide chain; the conserved Arg17 side chain interacts with the other oxygen atom of the alpha-carboxyl C terminus as well as with the side chain of Glu85. This is the first x-ray analysis of a protein of the phosphoenolpyruvate:sugar phosphotransferase system. Furthermore, this work represents a protein structure which has been solved by starting with a model that represented only one-third of the scattering matter.

Relationship between structure and antiviral activity of 5-methoxymethyl-2'-deoxyuridine and 5-methoxymethyl-1-(2'-deoxy-beta-D-lyxofuranosyl)uracil.

5-Methoxymethyl-1-(2'-deoxy-beta-D-lyxofuranosyl)uracil (MMdLU) was not active against the herpes simplex viruses. The relationship between molecular conformation and antiviral activity for the two epimers, 5-methoxymethyl-2'-deoxyuridine (MMdUrd) and MMdLU, is discussed. MMdUrd was phosphorylated by the virus-induced deoxythymidine kinase. In contrast, MMdLU did not serve as a substrate for the kinase. The geometry and distance between the 5'-CH2OH and 3'-OH groups of the furanose ring appear to be key factors in determining the efficiency of phosphorylation by the virus-induced deoxythymidine kinase, and hence antiviral activity.

Characterization of phosphorylated histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

The histidine-containing phosphocarrier protein (HPr) of the phosphoenolpyruvate:sugar phosphotransferase system, when phosphorylated, contains a 1-phosphohistidinyl (1-P-histidinyl) residue (His-15). The properties of this 1-P-histidinyl residue were investigated by using phospho-HPr (P-HPr), P-HPr-1, and P-HPr-2. HPr-1 and HPr-2 are deamidated forms of HPr produced by boiling. In addition, HPr-1 produced during frozen storage was investigated. Both pH and temperature dependencies of the rate of hydrolysis of the phosphoryl group of the 1-P-histidinyl residue were investigated. The results show that the 1-P-histidinyl residue in HPr and HPr-1 has significantly different properties from free 1-P-histidine and that these differences are attributable to the active-site residues Glu-66 and Arg-17 and the pK of the imidazole group of the 1-P-histidinyl residue in P-HPr. The 1-P-histidinyl residue in P-HPr and P-HPr-1 shows a greater lability at physiological pH than the free amino acid. A proposal for the active site of P-HPr is made on the basis of these results and the recently obtained tertiary structure. In contrast, the hydrolysis properties of the 1-P-histidinyl residue in P-HPr-2 were similar to those obtained for either free 1-P-histidine or denatured P-HPr. The loss of activity that is associated with boiling HPr was shown to be due to HPr-2 formation as HPr-1 was found to be fully active.